WO2010002277A1

WO2010002277A1 - Methods and compositions for improving trees

Info

Publication number: WO2010002277A1
Application number: PCT/NZ2009/000128
Authority: WO
Inventors: Sheree Alice Cato
Original assignee: New Zealand Forest Research Institute Limited
Priority date: 2008-06-30
Filing date: 2009-06-30
Publication date: 2010-01-07

Abstract

The invention provides a method for identifying a tree with a genotype indicative of at least one of increased wood density and increased cell wall thickness, the method including detecting in the tree, or a sample derived from the tree, by direct or indirect methods, the presence of: (i) a first allele of the dehydrin gene that encodes a dehydrin protein including a serine (S) at amino acid position (191) and a leucine (L) at amino acid position (212); and (ii) a second allele of the dehydrin gene that encodes a dehydrin protein including at least one of (a) a proline (P) at amino acid position (191), or (b) a valine (V) at amino acid position (212). The invention also provides the isolated polynucleotides of such alleles, constructs, host cells, plant cells and plants comprising such polynucleotides. The invention also provided methods for producing plants with at least one of increased wood density and increased cell wall thickness, using the polynucleotides of the invention, and plants produced by the methods.

Description

METHODS AND COMPOSITIONS FOR IMPROVING TREES

FIELD OF THE INVENTION

The present invention relates to methods and compositions for identifying or producing trees with at least one of increased wood density and increased cell wall thickness.

BACKGROUND

The wood density of trees is important in the forestry industry. Wood with increased density has improved strength and durability when used as sawn timber, and in furniture making. Wood density and cell wall thickness are also strongly correlated. Increased wood density/cell wall thickness lends to an increase in yield (per unit volume) which is beneficial for biomaterial/biofuel applications. For these and other applications timber from trees with increased wood density can be sold at a premium. It is therefore of significant interest and value to the forestry industry to adopt breeding strategies aimed at developing trees with increased wood density and/or cell wall thickness.

It is possible to measure wood density and/or cell wall thickness in mature trees, and to select trees with relatively increased wood density and/or cell wall thickness for use as parents in breeding programs designed to produce offspring with increased growth rate. However, measurement of wood density and/or cell wall thickness is time consuming and expensive. In addition the trees may need to reach relatively mature growth stage before useful wood density and/or cell wall thickness data can be collected.

Marker assisted selection (MAS) is an approacruthat is often used to identify plants or animals with alteration in a particular trait using a genetic marker associated with the trait. The alteration in the trait may be desirable and be advantageously selected for, or non-desirable and advantageously selected against, in selective breeding programs. MAS allows breeders to identify and select plants or animals at a relatively immature growth stage, and is particularly valuable for traits that are not revealed until the plant or animal reaches advanced maturity. The best markers for MAS are the causal polymorphisms or mutations, but where these are not available, markers that are linked, and preferably in linkage disequilibrium, with the causal mutation can also be used. Such informationcan be used to accelerate genetic gain, or reduce trait measurement costs, and thereby has utility in commercial breeding programmes.

To apply such approaches to improving wood density and/or cell wall thickness in trees, of course requires the availability of markers linked to the wood density and/or cell wall thickness trait. It would therefore be beneficial to have available markers that could be used to identify trees with increased wood density and/or cell wall thickness.

Advances in genetic manipulation provide the tools to transform plants, including trees, to contain and express foreign genes. This has led to the development of plants capable of expressing pharmaceuticals and other chemicals, plants with increased pest resistance, increased stress tolerance and many other beneficial traits. To use such approaches for increasing wood density and/or cell wall thickness in trees, it is necessary to identify genes that can influence wood density and/or cell wall thickness when introduced into trees by the genetic manipulation techniques.

It is an object of the invention to provide methods for identifying or producing trees with at least one of increased wood density and increased cell wall thickness, and/or at least to provide the public with a useful choice.

SUMMARY OF THE INVENTION

The present invention results from the applicants' discovery that two particular pairs of alleles of the dehydrin gene, when present in the heterozygous state, are associated with increased wood density and cell wall thickness. The first allele includes a serine (S) at amino acid position 191 and a leucine at amino acid position 212. The second allele includes at least one of (i) a proline at amino acid position 191, or (ii) a valine at amino acid position 212.

The invention provides methods for identifying and selecting trees with genotypes indicative of at least one of (i) increased wood density and (ii) increased cell wall thickness based on detection of presence of the first and second alleles in the heterozygous state. The invention also provides transgenic methods for producing trees with increased wood density and/or cell wall thickness by manipulating expression of these dehydrin alleles in trees.

In the first aspect the invention provides a method for identifying a tree with a genotype indicative of at least one of increased wood density and increased cell wall thickness, the method including detecting in the tree, or a sample derived from the tree, by direct or indirect methods, the presence of:

(i) a first allele of the dehydrin gene that includes a serine (S) at amino acid position 191 and a leucine (L) at amino acid position 212; and (ii) a second allele of the dehydrin gene that includes at least one of

(a) a proline (P) at amino acid position 191, or

(b) a valine (V) at amino acid position 212.

First dehydrin allele polypeptides - serine/leucine (S/L) allele polypeptides

Preferably the polypeptide of the first dehydrin allele, comprises a sequence with at least 70% identity to the polypeptide sequence of any one of SEQ ID NO: 13, 16, 17 and 20.

Preferably the polypeptide of the first dehydrin allele polypeptide has the sequence of any one of SEQ ID NO: 13, 16, 17 and 20.

First dehydrin allele polynucleotides - serine/leucine (S/L) allele polynucleotides

In one embodiment of the method of the invention the polynucleotide of the first dehydrin allele comprises a sequence with at least 70% identity to the polynucleotide sequence of any one of SEQ ID NO: 47, 50, 51 and 54.

Preferably the polynucleotide of the first dehydrin allele comprises the polynucleotide sequence of any one of SEQ ID NO: 47, 50, 51 and 54.

Preferably the polynucleotide contains a codon at nucleotide positions 571-573 encoding the serine (S) at amino acid position 191. Preferably the codon is TCT. Preferably the polynucleotide contains T at nucleotide position 571. Preferably the polynucleotide contains a codon at nucleotide positions 634-636 encoding the leucine (L) at amino acid position 212. Preferably the codon is CTT. Preferably the polynucleotide contains C at nucleotide position 634.

Second dehydrin allele -proline (P) allele polypeptides

In one embodiment the second dehydrin allele polypeptide comprises proline (P) at amino acid position i91.

Preferably the polypeptide of the second dehydrin allele polypeptide, comprises a sequence with at least 70% identity to the polypeptide sequence of any one of SEQ ID NO: 1-12 and 22-34.

Preferably the polypeptide of the first dehydrin allele polypeptide has the sequence of any one of SEQ ID NO: 1-12 and 22-34.

Second dehydrin allele -proline (P) allele polynucleotides

In one embodiment the second dehydrin allele polynucleotide includes a codon encoding a proline (P) residue at amino acid position 191.

Preferably the second dehydrin allele comprises a sequence with at least 70% identity to the polynucleotide sequence of any one of SEQ ID NO: 35-46 and 56-68.

Preferably the polynucleotide of the second dehydrin allele comprises the polynucleotide sequence of any one of SEQ ID NO: 35-46 and 56-68.

Preferably the polynucleotide contains a codon at nucleotide positions 571-573 encoding the proline (P) at amino acid position 191. Preferably the codon is CCT. Preferably the polynucleotide contains C at nucleotide position 571. Second dehydrin allele - valine (V) allele polypeptides

In a further embodiment the second dehydrin allele polypeptide comprises valine (V) at amino acid position 212.

Preferably the polypeptide of the second dehydrin allele polypeptide, comprises a sequence with at least 70% identity to the polypeptide sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.

Preferably the polypeptide of the second dehydrin allele polypeptide has the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.

Second dehydrin allele - valine (V) allele polynucleotides

In one embodiment the second dehydrin allele polynucleotide includes a codon encoding a valine (V) at amino acid position 212.

Preferably the second dehydrin allele comprises a sequence with at least 70% identity to the polynucleotide sequence of any one of SEQ ID NO: 48, 49, 52, 53 and 55.

Preferably the polynucleotide of the second dehydrin allele comprises the polynucleotide sequence of any one of SEQ ID NO: 48, 49, 52, 53 and 55.

Preferably the polynucleotide contains a codon at nucleotide positions 634-636 encoding the valine (V) at amino acid position 212. Preferably the codon is GTT. Preferably the polynucleotide contains G at nucleotide position 634.

Preferably the presence of the pair of heterozygous alleles is in LD with the increased wood density and/or cell wall thickness trait.

More preferably the presence of the pair of alleles is in LD with the increased wood density and/or cell wall thickness trait at a D' value of at least 0.1, more preferably at least 0.2, more preferably at least 0.3, more preferably at least 0.4, more preferably at least 0.5. More preferably the presence of the pair of alleles, or each haplotype, is in LD with the increased wood density/or cell wall thickness trait at a R² value of at least 0.05, more preferably at least 0.075, more preferably at least 0.1, more preferably at least 0.2, more preferably at least 0.3, more preferably at least 0.4, more preferably at least 0.5.

Presence of the first and/or second allele may be detected directly, or may be detected indirectly by detecting a marker that is linked to the first and/or second allele.

Preferably the marker is in linkage disequilibrium (LD) with the first or second allele.

Preferably the marker is in LD with the allele at a D' value of at least 0.1, more preferably at least 0.2, more preferably at least 0.3, more preferably at least 0.4, more preferably at least 0.5.

Preferably the marker is in LD with the allele at a R² value of at least 0.05, more preferably at least 0.075, more preferably at least 0.1, more preferably at least 0.2, more preferably at least 0.3, more preferably at least 0.4, more preferably at least 0.5.

Table 1 : Markers that are in linkage disequilibrium with one of the alleles, or codon encoding the characteristic amino acid in the dehydrin gene.

It will be appreciated by those skilled in the art that the protein isoforms are encoded by corresponding nucleic acid alleles. Thus the method of the invention can be applied by detecting the presence of the specified nucleotides in the polynucleotides encoding the allelic polypeptides, or by detecting the presence of the specified amino acids in the encoded allelic polypeptides.

The nucleic acid alleles, or linked nucleic acid markers, may be detected by any suitable method. Preferably the alleles or markers are detected using a polymerase chain reaction (PCR) step. PCR methods are well known to those skilled in the art and are described for example in Mullis et ah, Eds. 1994 The Polymerase Chain Reaction, Birkhauser, incorporated herein by reference.

Preferably a PCR product is produced by amplifying the marker with primers comprising sequence complimentary to sequence of the tree genome flanking the polymorphism or marker.

Any suitable primer pair may be used. Preferably the PCR is performed using at least one primer selected from those set forth in Table 2 Preferably the PCR is performed using at least one primer pair selected from those set forth in Table 2

Table 2: Primers for amplifying PCR products comprising the characteristic nucleotides of the dehydrin alleles invention

Other methods for detecting the presence of nucleotides characteristic of a specific allele are also contemplated, such as but not limited to probe-based methods, which are well known to those skilled in the art as described in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987, incorporated herein by reference.

PCR Products can also be sequence directly in order to identify S/L; P or V dehydrin alleles/ individuals. Use of other methods such as the oligonucleotide ligation assay (OLA) are also included within the scope of the invention. OLA methods are well known to those skilled in the art.

In. one embodiment presence of the first and/or second allele is detected directly by detecting the amino acid sequence difference that distinguishes each allele.

The presence of amino acids characteristic of specified alleles may also be detected in a protein, or polypeptide, sample derived from the tree. Any suitable method for detecting the presence of the characteristic amino acid in a protein or polypeptide may be applied. Typical methods involve the use of antibodies for detection of the protein polymorphism. Methods for producing and using antibodies are well known to those skilled in the art and are described for example in Antibodies, A Laboratory Manual, Harlow A Lane, Eds, Cold Spring Harbour Laboratory, 1998.

Selection method

In a further aspect the invention provides a method for selecting a tree with a genotype indicative of increased wood density and/or cell wall thickness, the method comprising selecting a tree identified by a method of the invention.

Transgenic methods

In a further aspect the invention provides a method for producing a tree cell or tree with at least one of increased wood density and increased cell wall thickness, the method comprising transformation of a tree cell or tree with a polynucleotide encoding a dehydrin polypeptide allele to produce a tree cell or tree with the heterozygous pair of alleles of the invention, shown to be linked to increased wood density and/or cell wall thickness.

In one embodiment the tree cell or tree is transformed to express an S/L dehydrin allele polypeptide.

In a further embodiment the tree cell or tree is transformed to express a P dehydrin allele polypeptide. In a further embodiment the tree cell or tree is transformed to express a V dehydrin allele polypeptide.

It will be understood by those skilled in the art that the polynucleotide allele transformed will depend on the polynucleotide endogenously expressed in the tree or tree cell or expressed in the tree cell or tree on three transgenic methods. The desired resulting heterologous combination of dehydrin alleles is at least one S/L allele and at least one P or V allele.

Serine/ Leucine (S/L) dehydrin allele polynucleotides transformed

In one embodiment the polynucleotide transformed encodes a dehydrin protein including a serine (S) residue at amino acid position 191 and a leucine (L) residue at amino acid position 212.

Preferably the dehydrin protein comprises a sequence with at least 70% identity to the polypeptide sequence of any one of SEQ ID NO: 13, 16, 17 and 20.

Preferably the dehydrin protein comprises the sequence of any one of SEQ ID NO: 13, 16, 17 and 20.

Preferably the polynucleotide transformed comprises a sequence with at least 70% identity to the polynucleotide sequence of any one of SEQ ID NO: 47, 50, 51 or 54.

Preferably the polynucleotide comprises the polynucleotide sequence of any one of SEQ ID NO: 47, 50, 51 or 54.

In this embodiment the second dehydrin polypeptide that the tree or tree cell comprises is a P allele or a V allele, so that the transformed tree cell or tree will ultimately contain one of the heterozygous combinations (S/L allele plus P allele or V allele) of dehydrin alleles that the applicants have shown are linked to wood density and cell wall thickness. Proline (P) dehydrin allele polynucleotides transformed

In one embodiment the polynucleotide transformed encodes a dehydrin protein including a proline (P) residue at amino acid position 191.

Preferably the dehydrin protein comprises a sequence with at least 70% identity to the polypeptide sequence of any one of SEQ ID NO: 1-12 and 22-23.

Preferably the dehydrin protein comprises the sequence of any one of SEQ ID NO: 1-12 and 22- 23.

Preferably the polynucleotide transformed comprises a sequence with at least 70% identity to the polynucleotide sequence of any one of SEQ ID NO: 35-46 and 56-58.

Preferably the polynucleotide comprises the polynucleotide sequence of any one of SEQ ID NO: 35-46 and 56-58.

In this embodiment the second dehydrin polypeptide that the tree or tree cell comprises is a serine/leucine (S/L) allele, so that the transformed tree cell or tree will ultimately contain one of the heterozygous combinations (S/L allele plus P allele) of dehydrin alleles that the applicants have shown are linked to wood density and cell wall thickness.

Proline (V) dehydrin allele polynucleotides transformed

In one embodiment the polynucleotide transformed encodes a dehydrin protein including a valine (V) residue at amino acid position 212.

Preferably the dehydrin protein comprises a sequence with at least 70% identity to the polypeptide sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.

Preferably the dehydrin protein comprises the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21. Preferably the polynucleotide transformed comprises a sequence with at least 70% identity to the polynucleotide sequence of any one of SEQ ID NO: 48, 49, 52, 53 and 55.

Preferably the polynucleotide comprises the polynucleotide sequence of any one of SEQ ID NO: 48, 49, 52, 53 and 55.

In this embodiment the second dehydrin polypeptide that the tree or tree cell comprises is a serine/leucine (S/L) allele, so that the transformed tree cell or tree will ultimately contain one of the heterozygous combinations (S/L allele plus V allele) of dehydrin alleles that the applicants have shown are linked to wood density and cell wall thickness.

In a further aspect the invention provides a tree cell or tree produced by a method of the invention.

Serine/Leucine (S/L) allele polynucleotides encoding polypeptides

In a further aspect the invention provides an isolated polynucleotide encoding a polypeptide with the sequence of any one of SEQ ID NO: 13, 16, 17 and 20 or a variant thereof, wherein the variant is a polypeptide capable of increasing at least one of wood density and cell wall thickness, when expressed in a plant.

In one embodiment the polypeptide has at least 70% identity to the sequence of any one of SEQ ID NO: 13, 16, 17 and 20.

Preferably the polypeptide includes a serine (S) residue at amino acid position 191 and a leucine residue at amino acid position 212.

Preferably the polypeptide comprises the sequence of any one of SEQ ID NO: 13, 16, 17 and 20.

Preferably the polypeptide consists of the sequence of any one of SEQ ID NO: 13, 16, 17 and 20. Serine/Leucine (S/L) allele polynucleotides

In a further aspect the invention provides an isolated polynucleotide comprising the sequence of any one of SEQ ID NO: 47, 50, 51 or 54 or a variant thereof, wherein the variant encodes a polypeptide capable of increasing at least one of wood density and cell wall thickness, when expressed in a plant.

In one embodiment the polynucleotide comprises a sequence with at least 70% identity to the polynucleotide sequence of any one of SEQ ID NO: 47, 50, 51 or 54.

In a further embodiment the polynucleotide includes a codon encoding serine (S) at nucleotide positions 571-573 and a codon encoding leucine (L) at nucleotide positions 634-636. Preferably the codon at nucleotide position 571-573 is TCT. Preferably the codon at nucleotide position

634-636 is CTT.

Preferably the polynucleotide comprises the sequence of any one of SEQ ID NO: 47, 50, 51 or

54.

Preferably the polynucleotide consists of the sequence of any one of SEQ ID NO: 47, 50, 51 or 54.

Serine/Leucine (S/L) allele polypeptides

In a further aspect the invention provides an isolated polypeptide with the sequence of any one of SEQ ID NO: 13, 16, 17 and 20 or a variant thereof, wherein the variant is a polypeptide capable of increasing at least one of wood density and cell wall thickness, when expressed in a plant.

Preferably the polypeptide includes a serine (S) residue at amino acid position 191 and a leucine (L) residue at amino acid position 212. Preferably the polypeptide comprises the sequence of any one of SEQ ID NO: 13, 16, 17 and 20.

Preferably the polypeptide consists of the sequence of any one of SEQ ID NO: 13, 16, 17 and 20.

Proline (P) allele polynucleotides encoding polypeptides

In a further aspect the invention provides an isolated polynucleotide encoding a polypeptide with the sequence of any one of SEQ ID NO: 1-12 and 22-34 or a variant thereof, wherein the variant is a polypeptide capable of increasing at least one of wood density and cell wall thickness, when expressed in a plant.

In one embodiment the polypeptide has at least 70% identity to the sequence of any one of SEQ ID NO: 1-12 and 22-34.

Preferably the polypeptide includes a proline (P) residue at amino acid position 191.

Preferably the polypeptide comprises the sequence of any one of SEQ ID NO: 1-12 and 22-34.

Preferably the polypeptide consists of the sequence of any one of SEQ ID NO: 1-12 and 22-34.

Proline (P) allele polynucleotides

In a further aspect the invention provides an isolated polynucleotide comprising the sequence of any one of SEQ ID NO: 35-46 and 56-68 or a variant thereof, wherein the variant encodes a polypeptide capable of increasing at least one of wood density and cell wall thickness, when expressed in a plant.

In one embodiment the polynucleotide comprises a sequence with at least 70% identity to the polynucleotide sequence of any one of SEQ ID NO: 35-46 and 56-68.

In a further embodiment the polynucleotide includes a codon encoding proline (P) at nucleotide positions 571-573. Preferably the codon at nucleotide position 571-573 is CCT. Preferably the polynucleotide comprises the sequence of any one of SEQ ID NO: 35-46 and 56- 68.

Preferably the polynucleotide consists of the sequence of any one of SEQ ID NO: 35-46 and 56- 68.

Proline (P) allele polypeptides

In a further aspect the invention provides an isolated polypeptide with the sequence of any one of SEQ ID NO: 1-12 and 22-34 or a variant thereof, wherein the variant is a polypeptide capable of increasing at least one of wood density and cell wall thickness, when expressed in a plant.

Preferably the polypeptide includes a serine (P) residue at amino acid position 191.

Valine (V) allele polynucleotides encoding polypeptides

In a further aspect the invention provides an isolated polynucleotide encoding a polypeptide with the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21 or a variant thereof, wherein the variant is a polypeptide capable of increasing at least one of wood density and cell wall thickness, when expressed in a plant.

In one embodiment the polypeptide has at least 70% identity to the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.

Preferably the polypeptide includes a valine (V) residue at amino acid position 212. Preferably the polypeptide comprises the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.

Preferably the polypeptide consists of the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.

Valine (V) allele polynucleotides

In a further aspect the invention provides an isolated polynucleotide comprising the sequence of any one of SEQ ID NO: 48, 49, 52, 53 and 55 or a variant thereof, wherein the variant encodes a polypeptide capable of increasing at least one of wood density and cell wall thickness, when expressed in a plant.

In one embodiment the polynucleotide comprises a sequence with at least 70% identity to the polynucleotide sequence of any one of SEQ ID NO: 48, 49, 52, 53 and 55.

In a further embodiment the polynucleotide includes a codon encoding valine (V) at nucleotide positions 634-636. Preferably the codon at nucleotide position 634-636 is GTT.

Preferably the polynucleotide comprises the sequence of any one of SEQ ID NO: 48, 49, 52, 53 and 55.

Preferably the polynucleotide consists of the sequence of any one of SEQ ID NO: 48, 49, 52, 53 and 55.

Valine (S/L) allele polypeptides

In a further aspect the invention provides an isolated polypeptide with the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21 or a variant thereof, wherein the variant is a polypeptide capable of increasing at least one of wood density and cell wall thickness, when expressed in a plant. In one embodiment the polypeptide has at least 70% identity to the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.

Preferably the polypeptide includes a valine (V) residue at amino acid position 212.

Preferably the polypeptide comprises the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.

In a further aspect the invention provides a polynucleotide encoding a polypeptide of the invention.

Constructs

In a further aspect the invention provides a genetic construct comprising a polynucleotide of the invention.

In one embodiment the genetic construct is an expression construct.

In a further aspect the invention provides a vector comprising a polynucleotide, genetic construct or expression construct of the invention.

In a further aspect the invention provides a host cell comprising a polynucleotide, genetic construct or expression construct of the invention.

In a further aspect the invention provides a host cell genetically modified to express a polynucleotide of the invention.

In a further aspect the invention provides a plant cell comprising a genetic construct or the expression construct of the invention. In a further aspect the invention provides a plant cell genetically modified to express a polynucleotide of the invention.

In a further aspect the invention provides a plant which comprises a plant cell of the invention.

Preferably the plants of the invention are trees, and the plant cells are tree cells.

In a further aspect the invention provides a group of trees selected by a method of the invention.

Trees

The trees in the methods of the invention may be from any tree species.

Preferred trees are those from gymnosperm species such as, but not limited to: Abies amabilis, Abies balsamea, Abies concolor, Abies grandis, Abies lasiocarpa, Abies magnified, Abies procera, Chamaecyparis lawsoniona, Chamaecyparis nootkatensis, Chamaecyparis thyoides, Juniperus virginiana, Larix decidua, Larix laricina, Larix leptolepis, Larix occidentalis, Larix siberica, Libocedrus decurrens, Picea abies, Picea engelmanni, Picea glauca, Picea mariana, Picea pungens, Picea rubens, Picea sitchensis, Pinus banksiana, Pinus brutia, Pinus caribaea, Pinus clausa, Pinus contorta, Pinus coulteri, Pinus echinata, Pinus eldarica, Pinus ellioti, Pinus jejfreyi, Pinus lambertiana, Pinus monticola, Pinus nigra, Pinus palustrus, Pinus pinaster, Pinus ponderosa, Pinus radiata, Pinus resinosa, Pinus rigida, Pinus serotina, Pinus strobus, Pinus sylvestris, Pinus taeda, Pinus virginiana; Pseudotsuga menziesii, Sequoia gigantea, Sequoia sempervirens, Taxodium distichum, Tsuga canadensis, Tsuga heterophylla, Tsuga mertensiana, Thuja occidentalis, and Thuja plicata.

Particularly preferred trees are those of the Pinus genus, including but limited to: Pinus banksiana, Pinus brutia, Pinus caribaea, Pinus clausa, Pinus contorta, Pinus coulteri, Pinus echinata, Pinus eldarica, Pinus ellioti, Pinus Jeffrey i, Pinus lambertiana, Pinus monticola, Pinus nigra, Pinus palustrus, Pinus pinaster, Pinus ponderosa, Pinus radiata, Pinus resinosa, Pinus ήgida, Pinus serotina, Pinus strobus, Pinus sylvestris, Pinus taeda, and Pinus virginiana.

Preferred Pinus species are those from the subgenus Pinus subsection Trifolia. Preferred subsection Trifolia species include P. taeda, Pinus radiata, P. attenuate, P. muricata, P. teocote, P. greggiii, P. herrerae, P. devoniana, P. pseudostrobus, and P. contorta.

Preferred Pinus species also include those selected from the group including Pinus radiata, Pinus taeda, Pinus sylvestris and Pinus pinaster.

Particularly preferred Pinus species include Pinus radiata and Pinus taeda.

A particularly preferred Pinus species is Pinus radiata.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

The term "comprising" as used in this specification means "consisting at least in part of. When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.

The term "dehydrin" in relation to polypeptides, proteins, polynucleotides and genes has the same meaning as dehydrin as commonly used by those skilled in the art. Dehydrins (DHNs) are part of a large group of highly hydrophilic proteins known as LEA (Late

Embryogenesis Abundant). The distinctive feature of all DHNs is a conserved, lysine-rich 15- amino acid domain, EKKGIMDKIKEKLPG, named the K-segment. It is usually present near the C-terminus. Other typical dehydrin features are: a track of Ser residues (the S-segment); a consensus motif, T/VDEYGNP (the Y-segment), located near the N-terminus; and less conserved regions, usually rich in polar amino acids (the Phi-segments). The number and order of the Y-, S-and K-segments define different DHN sub-classes: Y(n)SK(n), Y(n)K(n), SK(n),

K(n) and K(n)S. (Rorat, T., 2006, Plant dehydrins— tissue location, structure and function., Cell MoI Biol Lett. 2006; 11(4):536-56. Epub 2006 Sept).

Dehydrins are distributed in a wide range of organisms including the higher plants, algae, yeast and cyanobacteria. They accumulate late in embryogenesis, and in nearly all the vegetative tissues during normal growth conditions and in response to stress leading to cellular dehydration (e.g. drought, low temperature and salinity). DHNs are localized in different cell compartments, such as the cytosol, nucleus, mitochondria, vacuole, and the vicinity of the plasma membrane; however, they are primarily localized to the cytoplasm and nucleus.

In vitro experiments have revealed that some DHNs (YSK(n)-type) bind to lipid vesicles that contain acidic phospholipids, and others (K(n)S) were shown to bind metals and have the ability to scavenge hydroxyl radicals [Asghar, R. et al. Protoplasma 177 (1994) 87-94], protect lipid membranes against peroxidation or display cryoprotective activity towards freezing-sensitive enzymes. The SK(n)-and K-type seem to be directly involved in cold acclimation processes. The main question arising from the in vitro findings is whether each DHN structural type could possess a specific function and tissue distribution. Much recent in vitro data clearly indicates that dehydrins belonging to different subclasses exhibit distinct functions.

"Wood density" as used herein means a measure of wood mass relative to wood volume and is often defined with the units kilogram per cubic metre kgm^" .

Wood density can be measured by several methods well known to those skilled in the art. Such methods include using x-ray densitometry (Cown et α/.,1983, Wood Sci. Technol. 17, 91) , the maximum moisture content method (Smith, 1954, Forest Products Laboratory Report No. 2014. Wisconsin, US Forest Service), and SilviScan (Evans et ai, 1995, Appita J. 48, 134).

"Cell wall thickness" as used herein means the distance across the cell wall from the lumen to the exterior of the cell. Cell wall thickness is usually measured in micrometers (μm).

Cell wall thickness can be measured by several methods well known to those skilled in the art. Such methods include using x-ray densitometry (Cown et α/.,1983, Wood Sci. Technol. 17, 91) and SilviScan (Evans et al, 1995, Appita J. 48, 134).

Methods for measuring wood density and cell wall thickness are also provided in the examples section of this specification.

"Polymorphism" is a condition in DNA in which the most frequent variant (or allele) has a population frequency which does not exceed 99%.

The term "linkage disequilibrium" or LD as used herein, refers to a derived statistical measure of the strength of the association or co-occurrence of two independent genetic markers. Various statistical methods can be used to summarize linkage disequilibrium (LD) between two markers but in practice only two, termed D' and R², are widely used.

Marker linked, and or in LD, with the specified polymorphisms may be of any type including but not limited to, SNPs, substitutions, insertions, deletions, indels, and simple sequence repeats (SSRs).

The abbreviation "SSR" stands for a "simple sequence repeat" and refers to any short sequence, for example, a mono-, di-, tri-, or tetra-nucleotide that is repeated at least once in a particular nucleotide sequence. These sequences are also known in the art as "microsatellites." A SSR can be represented by the general formula (Nl N2 . . . Ni)n, wherein N represents nucleotides A, T, C or G, i represents the number of the nucleotides in the base repeat, and n represents the number of times the base is repeated in a particular DNA sequence. The base repeat, i.e., Nl N2 . . . Ni, is also referred to herein as an "SSR motif." For example, (ATC)4, refers to a tri-nucleotide ATC motif that is repeated four times in a particular sequence. In other words, (ATC)4 is a shorthand version of "ATCATCATCATC."

The term "complement of a SSR motif refers to a complementary strand of the represented motif. For example, the complement of (ATG) motif is (TAC).

The term "SSR locus" refers to a location on a chromosome of a SSR motif; locus may be occupied by any one of the alleles of the repeated motif. "Allele" is one of several alternative forms of the SSR motif occupying a given locus on the chromosome. For example, the (ATC)8 locus refers to the fragment of the chromosome containing this repeat, while (ATC)4 and (ATC)7 repeats represent two different alleles of the (ATC)8 locus. As used herein, the term locus refers to the repeated SSR motif and the flanking 5' and 3' non-repeated sequences. SSR loci of the invention are useful as genetic markers, such as for determination of polymorphism.

The terms "tree", "tree plant" and "plants" can be used interchangeably throughout this specification.

The term "polynucleotide(s)," as used herein, means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 15 nucleotides, and include as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polynucleotides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes or primers and fragments.

The term "primer" refers to a short polynucleotide, usually having a free 3 'OH group, that is hybridized to a template and used for priming polymerization of a polynucleotide complementary to the target.

The term "probe" refers to a short polynucleotide that is used to detect a polynucleotide sequence, that is complementary to the probe, in a hybridization-based assay. Polynucleotides and fragments

The term "polynucleotide(s)," as used herein, means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 15 nucleotides, and include as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments.

A "fragment" of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides that is capable of specific hybridization to a target of interest, e.g., a sequence that is at least 15 nucleotides in length. The fragments of the invention comprise 15 nucleotides, preferably at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 50 nucleotides, more preferably at least 50 nucleotides and most preferably at least 60 nucleotides of contiguous nucleotides of a polynucleotide of the invention. A fragment of a polynucleotide sequence can be used in antisense, gene silencing, triple helix or ribozyme technology, or as a primer, a probe, included in a microarray, or used in polynucleotide-based selection methods of the invention.

The term "probe" refers to a short polynucleotide that is used to detect a polynucleotide sequence, that is complementary to the probe, in a hybridization-based assay. The probe may consist of a "fragment" of a polynucleotide as defined herein.

Polypeptides and fragments

The term "polypeptide", as used herein, encompasses amino acid chains of any length but preferably at least 5 amino acids, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds. Polypeptides of the present invention may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof.

A "fragment" of a polypeptide is a subsequence of the polypeptide that performs a function that is required for the biological activity and/or provides three dimensional structure of the polypeptide. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof capable of performing the above enzymatic activity.

The term "isolated" as applied to the polynucleotide or polypeptide sequences disclosed herein is used to refer to sequences that are removed from their natural cellular environment. An isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques.

The term "recombinant" refers to a polynucleotide sequence that is removed from sequences that surround it in its natural context and/or is recombined with sequences that are not present in its natural context.

A "recombinant" polypeptide sequence is produced by translation from a "recombinant" polynucleotide sequence.

The term "derived from" with respect to polynucleotides and polypeptides of the invention being "derived from" a particular genera or species, means that the polynucleotide or polypeptide has the same sequence as a polynucleotide or polypeptide found naturally in that genera or species. The polynucleotide or polypeptide which is derived from a genera or species may therefore be produced synthetically or recombinantly.

Variants

As used herein, the term "variant" refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the inventive polypeptides and polypeptides possess biological activities that are the same or similar to those of the inventive polypeptides or polypeptides. The term "variant" with reference to polypeptides and polypeptides encompasses all forms of polypeptides and polypeptides as defined herein.

Polynucleotide variants

Variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably_at least 76%;^* more preferably at least %, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a specified polynucleotide sequence. Identity is found over a comparison window of at least 20 nucleotide positions, preferably at least 50 nucleotide positions, more preferably at least 100 nucleotide positions, and most preferably over the entire length of the specified polynucleotide sequence. Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in b!2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.

The identity of polynucleotide sequences may be examined using the following unix command line parameters:

bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p blastn

The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. The bl2seq program reports sequence identity as both the number and percentage of identical nucleotides in a line "Identities = ".

Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (e.g. Needleman, S. B. and Wunsch, C. D. (1970) J. MoI. Biol. 48, 443-453). A full implementation of the Needleman- Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice,P. LongdenJ. and Bleasby,A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp.276- 277) which can be obtained from http://www.hgmp.mrc.ac.uk/Software/EMBOSS/. The European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at http:/www.ebi. ac.uk/emboss/align/.

Alternatively the GAP program may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences

10, 227-235. Use of BLASTN as described above is preferred for use in the determination of sequence identity for polynucleotide variants according to the present invention.

Polynucleotide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/).

The similarity of polynucleotide sequences may be examined using the following unix command line parameters:

bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p tblastx

The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. The size of this database is set by default in the bl2seq program. For small E values, much less than one, the E value is approximately the probability of such a random match.

Variant polynucleotide sequences preferably exhibit an E value of less than 1 x 10 ^"10 more preferably less than 1 x 10 ^"20, more preferably less than 1 x 10 ³⁰, more preferably less than 1 x 10 ^"40, more preferably less than 1 x 10 ^"5O _s more preferably less than 1 x 10 ^"60 _> more preferably less than 1 x 10 ^"70 _; more preferably less than 1 x 10 ^"8^ more preferably less than 1 x 10 ^"90 and most preferably less than 1 x 10 ^"l0° when compared with any one of the specifically identified sequences.

Alternatively, variant polynucleotides of the present invention hybridize to a specified polynucleotide sequence, or complements thereof under stringent conditions. The term "hybridize under stringent conditions", and grammatical equivalents thereof, refers to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration. The ability to hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired stringency.

With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30° C (for example, 10° C) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al, Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al, 1987, Current Protocols in Molecular Biology, Greene Publishing,). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm = 81. 5 + 0. 41% (G + C-log (Na+). (Sambrook et al, Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84:1390). Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65⁰C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65⁰ C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65⁰C.

With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 10° C below Tm. On average, the Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length)⁰ C.

With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen et al., Science. 1991 Dec 6;254(5037): 1497-500) Tm values are higher than those for DNA-DNA or DNA-RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov l;26(21):5004-6. Exemplary stringent hybridization conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10° C below the Tm.

Variant polynucleotides of the present invention also encompasses polynucleotides that differ from the sequences of the invention but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention. A sequence alteration that does not change the amino acid sequence of the polypeptide is a "silent variation". Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism.

Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al, 1990, Science 247, 1306).

Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/) via the tblastx algorithm as previously described.

Polypeptide Variants

The term "variant" with reference to polypeptides encompasses naturally occurring, recombinantly and synthetically produced polypeptides. Variant polypeptide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least %, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a sequences of the present invention. Identity is found over a comparison window of at least 20 amino acid positions, preferably at least 50 amino acid positions, more preferably at least 100 amino acid positions, and most preferably over the entire length of a polypeptide of the invention.

Polypeptide sequence identity can be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq, which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off.

Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available at http:/www.ebi.ac.uk/emboss/align/) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227- 235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity.

Use of BLASTP as described above is preferred for use in the determination of polypeptide variants according to the present invention.

Polypeptide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The similarity of polypeptide sequences may be examined using the following unix command line parameters: bl2seq -i peptideseql -j peptideseq2 -F F -p blastp

Variant polypeptide sequences preferably exhibit an E value of less than 1 x 10 ^"10 more preferably less than 1 x 10 ^"20, more preferably less than 1 x 10 ^"30, more preferably less than 1 x 10 ^⁰, more preferably less than 1 x 10 ^"5^ more preferably less than 1 x 10 ^"6^ more preferably less than 1 x 10 ^~7 , more preferably less than 1 x 10 ^"8^ more preferably less than 1 x 10 ^"90 and most preferably less than 1 x 10 ^"10° when compared with any one of the specifically identified sequences.

The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match.

Conservative substitutions of one or several amino acids of a described polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al, 1990, Science 247, 1306).

Constructs, vectors and components thereof

The term "genetic construct" refers to a polynucleotide molecule, usually double-stranded DNA, which may have inserted into it another polynucleotide molecule (the insert polynucleotide molecule) such as, but not limited to, a cDNA molecule. A genetic construct may contain the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. The insert polynucleotide molecule may be derived from the host cell, or may be derived from a different cell or organism and/or may be a recombinant polynucleotide. Once inside the host cell the genetic construct may become integrated in the host chromosomal DNA. The genetic construct may be linked to a vector. The term "vector" refers to a polynucleotide molecule, usually double stranded DNA, which is used to transport the genetic construct into a host cell. The vector may be capable of replication in at least one additional host system, such as E. coli.

The term "expression construct" refers to a genetic construct that includes the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. An expression construct typically comprises in a 5' to 3' direction: a) a promoter functional in the host cell into which the construct will be transformed, b) the polynucleotide to be expressed, and c) a terminator functional in the host cell into which the construct will be transformed.

The term "coding region" or "open reading frame" (ORF) refers to the sense strand of a genomic DNA sequence or a cDNA sequence that is capable of producing a transcription product and/or a polypeptide under the control of appropriate regulatory sequences. The coding sequence is identified by the presence of a 5' translation start codon and a 3' translation stop codon. When inserted into a genetic construct, a "coding sequence" is capable of being expressed when it is operably linked to promoter and terminator sequences.

"Operably-linked" means that the sequenced to be expressed is placed under the control of regulatory elements that include promoters, tissue-specific regulatory elements, temporal regulatory elements, enhancers, repressors and terminators.

The term "noncoding region" refers to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site. These sequences are also referred to respectively as the 5' UTR and the 3' UTR. These regions include elements required for transcription initiation and termination and for regulation of translation efficiency.

Terminators are sequences, which terminate transcription, and are found in the 3' untranslated ends of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulatory functions. The term "promoter" refers to nontranscribed cis-regulatory elements upstream of the coding region that regulate gene transcription. Promoters comprise cis-initiator elements which specify the transcription initiation site and conserved boxes such as the TATA box, and motifs that are bound by transcription factors.

A "transgene" is a polynucleotide that is taken from one organism and introduced into a different organism by transformation. The transgene may be derived from the same species or from a different species as the species of the organism into which the transgene is introduced.

An "inverted repeat" is a sequence that is repeated, where the second half of the repeat is in the complementary strand, e.g.,

(5')GATCTA TAGATC(3')

(3')CTAGAT ATCTAG(5')

Read-through transcription will produce a transcript that undergoes complementary base-pairing to form a hairpin structure provided that there is a 3-5 bp spacer between the repeated regions.

A "transgenic plant" refers to a plant which contains new genetic material as a result of genetic manipulation or transformation. The new genetic material may be derived from a plant of the same species as the resulting transgenic plant or from a different species.

The terms "to alter expression of and "altered expression" of a polynucleotide or polypeptide of the invention, are intended to encompass the situation where genomic DNA corresponding to a polynucleotide of the invention is modified thus leading to altered expression of a polynucleotide or polypeptide of the invention. Modification of the genomic DNA may be through genetic transformation or other methods known in the art for inducing mutations. The "altered expression" can be related to an increase or decrease in the amount of messenger RNA and/or polypeptide produced and may also result in altered activity of a polypeptide due to alterations in the sequence of a polynucleotide and polypeptide produced.

The invention provides methods for selecting and producing plants altered in wood density, relative to suitable control plants. Suitable control plants may include non-transformed plants of the same species and variety, or plants of the same species or variety transformed with a control construct.

Methods for isolating polynucleotides

The polynucleotide molecules of the invention can be isolated by using a variety of techniques known to those of ordinary skill in the art. By way of example, such polypeptides can be isolated through use of the polymerase chain reaction (PCR) described in Mullis et ah, Eds. 1994 The Polymerase Chain Reaction, Birkhauser, incorporated herein by reference. The polypeptides of the invention can be amplified using primers, as defined herein, derived from the polynucleotide sequences of the invention.

Further methods for isolating polynucleotides of the invention, or polynucleotides useful in methods of the invention, include use of all, or portions of, the polynucleotides set forth herein as hybridization probes. The technique of hybridizing labelled polynucleotide probes to polynucleotides immobilized on solid supports such as nitrocellulose filters or nylon membranes, can be used to screen the genomic or cDNA libraries. Exemplary hybridization and wash conditions are: hybridization for 20 hours at 65°C in 5. 0 X SSC, 0. 5% sodium dodecyl sulfate, 1 X Denhardt's solution ; washing (three washes of twenty minutes each at 55°C) in 1. 0 X SSC, 1% (w/v) sodium dodecyl sulfate, and optionally one wash (for twenty minutes) in 0. 5 X SSC, 1% (w/v) sodium dodecyl sulfate, at 60°C. An optional further wash (for twenty minutes) can be conducted under conditions of 0. 1 X SSC, 1% (w/v) sodium dodecyl sulfate, at 60°C.

The polynucleotide fragments of the invention may be produced by techniques well-known in the art such as restriction endonuclease digestion and oligonucleotide synthesis.

A partial polynucleotide sequence may be used, in methods well-known in the art to identify the corresponding full length polynucleotide sequence. Such methods include PCR-based methods, 5'RACE (Frohman MA, 1993, Methods Enzymol. 218: 340-56) and hybridization- based method, computer/database -based methods. Further, by way of example, inverse PCR permits acquisition of unknown sequences, flanking the polynucleotide sequences disclosed herein, starting with primers based on a known region (Triglia et al., 1998, Nucleic Acids Res 16, 8186, incorporated herein by reference). The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. Divergent primers are designed from the known region. In order to physically assemble full-length clones, standard molecular biology approaches can be utilized (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).

It may be beneficial, when producing a transgenic plant from a particular species, to transform such a plant with a sequence or sequences derived from that species. The benefit may be to alleviate public concerns regarding cross-species transformation in generating transgenic organisms. Additionally when down-regulation of a gene is the desired result, it may be necessary to utilise a sequence identical (or at least highly similar) to that in the plant, for which reduced expression is desired. For these reasons among others, it is desirable to be able to identify and isolate orthologues of a particular gene in several different plant species. Variants (including orthologues) may be identified by the methods described.

Methods for identifying variants

Physical methods

Variant polynucleotides may be identified using PCR-based methods (Mullis et al, Eds. 1994 The Polymerase Chain Reaction, Birkhauser). Typically, the polynucleotide sequence of a primer, useful to amplify variant polynucleotide molecules by PCR, may be based on a sequence encoding a conserved region of the corresponding amino acid sequence.

Alternatively library screening methods will be known to those skilled in the art (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) may be employed. When identifying variants of the probe sequence hybridisation and/or wash stringency conditions will typically be reduced relative to when exact sequence matches are sought.

Polypeptide variants of the invention may be identified by physical methods, for example by screening expression libraries using antibodies raised against polypeptides of the invention (Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) or by identifying polypeptides from natural sources with the aid of such antibodies.

Computer based methods

The variant sequences of the invention, including both polynucleotide and polypeptide variants, may also be identified by computer-based methods well-known to those skilled in the art, using public domain sequence alignment algorithms and sequence similarity search tools to search sequence databases (public domain databases include Genbank, EMBL, Swiss-Prot, PIR and others). See, e.g., Nucleic Acids Res. 29: 1-10 and 1 1-16, 2001 for examples of online resources. Similarity searches retrieve and align target sequences for comparison with a sequence to be analyzed (i.e., a query sequence). Sequence comparison algorithms use scoring matrices to assign an overall score to each of the alignments.

An exemplary family of programs useful for identifying variants in sequence databases is the BLAST suite of programs (version 2.2.5 [Nov 2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from (ftp://ftp.ncbi.nih.gov/blast/) or from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894 USA. The NCBI server also provides the facility to use the programs to screen a number of publicly available sequence databases. BLASTN compares a nucleotide query sequence against a nucleotide sequence database. BLASTP compares an amino acid query sequence against a protein sequence database. BLASTX compares a nucleotide query sequence translated in all reading frames against a protein sequence database. tBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames. tBLASTX compares the six- frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. The BLAST programs may be used with default parameters or the parameters may be altered as required to refine the screen.

The use of the BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX, is described in the publication of Altschul et al, Nucleic Acids Res. 25: 3389-3402, 1997. The "hits" to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, BLASTX, tBLASTN, tBLASTX, or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.

The BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also produce "Expect" values for alignments. The Expect value (E) indicates the number of hits one can "expect" to see by chance when searching a database of the same size containing random contiguous sequences. The Expect value is used as a significance threshold for determining whether the hit to a database indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the database screened, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in that database is 1% or less using the BLASTN, BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.

Multiple sequence alignments of a group of related sequences can be carried out with CLUSTALW (Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673- 4680, http://www-igbmc.u-strasbg.fr/BioInfo/ClustalW/Top.htmn or T-COFFEE (Cedric Notredame, Desmond G. Higgins, Jaap Heringa, T-Coffee: A novel method for fast and accurate multiple sequence alignment, J. MoI. Biol. (2000) 302: 205-217))or PILEUP, which uses progressive, pairwise alignments. (Feng and Doolittle, 1987, J. MoI. Evol. 25, 351).

Pattern recognition software applications are available for finding motifs or signature sequences. For example, MEME (Multiple Em for Motif Elicitation) finds motifs and signature sequences in a set of sequences, and MAST (Motif Alignment and Search Tool) uses these motifs to identify similar or the same motifs in query sequences. The MAST results are provided as a series of alignments with appropriate statistical data and a visual overview of the motifs found. MEME and MAST were developed at the University of California, San Diego. PROSITE (Bairoch and Bucher, 1994, Nucleic Acids Res. 22, 3583; Hofmann et al., 1999, Nucleic Acids Res. 27, 215) is a method of identifying the functions of uncharacterized proteins translated from genomic or cDNA sequences. The PROSITE database (www.expasy.org/prosite) contains biologically significant patterns and profiles and is designed so that it can be used with appropriate computational tools to assign a new sequence to a known family of proteins or to determine which known domain(s) are present in the sequence (Falquet et al., 2002, Nucleic Acids Res. 30, 235). Prosearch is a tool that can search SWISS-PROT and EMBL databases with a given sequence pattern or signature.

Methods for isolating polypeptides

The polypeptides of the invention, including variant polypeptides, may be prepared using peptide synthesis methods well known in the art such as direct peptide synthesis using solid phase techniques (e.g. Stewart et al., 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco California, or automated synthesis, for example using an Applied Biosystems 43 IA Peptide Synthesizer (Foster City, California). Mutated forms of the polypeptides may also be produced during such syntheses.

The polypeptides and variant polypeptides of the invention may also be purified from natural sources using a variety of techniques that are well known in the art (e.g. Deutscher, 1990, Ed, Methods in Enzymology, Vol. 182, Guide to Protein

Alternatively the polypeptides and variant polypeptides of the invention may be expressed recombinantly in suitable host cells and separated from the cells as discussed below.

Methods for producing constructs and vectors

The genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/or polynycleotides encoding polypeptides of the invention, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or plant organisms. The genetic constructs of the invention are intended to include expression constructs as herein defined. Methods for producing and using genetic constructs and vectors are well known in the art and are described generally in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing, 1987).

Methods for producing host cells comprising constructs and vectors

The invention provides a host cell which comprises a genetic construct or vector of the invention. Host cells may be derived from, for example, bacterial, fungal, insect, mammalian or plant organisms.

Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art (e.g. Sambrook et al, Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides of the invention. Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention. The expressed recombinant polypeptide, which may optionally be secreted into the culture, may then be separated from the medium, host cells or culture medium by methods well known in the art (e.g. Deutscher, Ed, 1990, Methods in Enzymology, VoI 182, Guide to Protein Purification).

Host cells of the invention may also be useful in methods for production of an enzymatic product generated by an expressed polypeptide of the invention. Such methods may involve culturing the host cells of the invention in a medium suitable for expression of a recombinant polypeptide of the invention, optionally in the presence of additional enzymatic substrate for the expressed polypeptide of the invention. The enzymatic product produced may then be separated from the host cells or medium by a variety of art standard methods.

Methods for producing plant cells and plants comprising constructs and vectors

The invention further provides plant cells. Production of these plants with altered wood density may be achieved through methods of the invention. Such methods may involve the transformation of these plant cells and plants, with a designed to alter expression of a polynucleotide or polypeptide capable of modulating wood density in such plant cells and plants. Such methods also include the transformation of plant cells and plants with a combination of the constructs designed to alter expression of one or more polypeptides or polypeptides capable of modulating wood density in such plant cells and plants.

Methods for transforming plant cells, plants and portions thereof with polynucleotides are described in Draper et al., 1988, Plant Genetic Transformation and Gene Expression. A Laboratory Manual Blackwell Sci. Pub. Oxford, p. 365; Potrykus and Spangenburg, 1995, Gene

Transfer to Plants. Springer- Verlag, Berlin.; and Gelvin et al, 1993, Plant Molecular Biol.

Manual. Kluwer Acad. Pub. Dordrecht. A review of transgenic plants, including transformation techniques, is provided in Galun and Breiman, 1997, Transgenic Plants. Imperial College Press,

London.

Methods for genetic manipulation of plants

A number of strategies for genetically manipulating plants are available (e.g. Birch, 1997, Ann Rev Plant Phys Plant MoI Biol, 48, 297). For example, strategies may be designed to increase expression of a polynucleotide/polypeptide in a plant cell, organ and/or at a particular developmental stage where/when it is normally expressed or to ectopically express a polynucleotide/polypeptide in a cell, tissue, organ and/or at a particular developmental stage which/when it is not normally expressed. The expressed polynucleotide/polypeptide may be derived from the plant species to be transformed or may be derived from a different plant species.

Transformation strategies may be designed to reduce expression of a polynucleotide/polypeptide in a plant cell, tissue, organ or at a particular developmental stage which/when it is normally expressed. Such strategies are known as gene silencing strategies.

Genetic constructs for expression of genes in transgenic plants typically include promoters for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detest presence of the genetic construct in the transformed plant. The promoters suitable for use in the constructs of this invention are functional in a cell, tissue or organ of a monocot or dicot plant and include cell-, tissue- and organ-specific promoters, cell cycle specific promoters, temporal promoters, inducible promoters, constitutive promoters that are active in most plant tissues, and recombinant promoters. Choice of promoter will depend upon the temporal and spatial expression of the cloned polynucleotide, so desired. The promoters may be those normally associated with a transgene of interest, or promoters which are derived from genes of other plants, viruses, and plant pathogenic bacteria and fungi. Those skilled in the art will, without undue experimentation, be able to select promoters that are suitable for use in modifying and modulating plant traits using genetic constructs comprising the polynucleotide sequences of the invention. Examples of constitutive plant promoters include the CaMV 35S promoter, the nopaline synthase promoter and the octopine synthase promoter, and the Ubi 1 promoter from maize. Plant promoters which are active in specific tissues, respond to internal developmental signals or external abiotic or biotic stresses are described in the scientific literature. Exemplary promoters are described, e.g., in WO 02/00894, which is herein incorporated by reference.

Exemplary terminators that are commonly used in plant transformation genetic construct include, e.g., the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zin gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solarium tuberosum PI-II terminator.

Selectable markers commonly used in plant transformation include the neomycin phophotransferase II gene (NPT II) which confers kanamycin resistance, the aadA gene, which confers spectinomycin and streptomycin resistance, the phosphinothricin acetyl transferase (bar gene) for Ignite (AgrEvo) and Basta (Hoechst) resistance, and the hygromycin phosphotransferase gene ( hpt) for hygromycin resistance.

Use of genetic constructs comprising reporter genes (coding sequences which express an activity that is foreign to the host, usually an enzymatic activity and/or a visible signal (e.g., luciferase,

GUS, GFP) which may be used for promoter expression analysis in plants and plant tissues are also contemplated. The reporter gene literature is reviewed in Herrera-Estrella et al., 1993, Nature 303, 209, and Schrott, 1995, In: Gene Transfer to Plants (Potrykus, T., Spangenbert. Eds) Springer Verlag. Berline, pp. 325-336.

Gene silencing strategies may be focused on the gene itself or regulatory elements which effect expression of the encoded polypeptide. "Regulatory elements" is used here in the widest possible sense and includes other genes which interact with the gene of interest.

Genetic constructs designed to decrease or silence the expression of a polynucleotide/polypeptide of the invention may include an antisense copy of a polynucleotide of the invention. In such constructs the polynucleotide is placed in an antisense orientation with respect to the promoter and terminator.

An "antisense" polynucleotide is obtained by inverting a polynucleotide or a segment of the polynucleotide so that the transcript produced will be complementary to the mRNA transcript of the gene, e.g.,

5'GATCTA 3' (coding strand) 3'CTAGAT 5' (antisense strand)

3'CUAGAU 5' mRNA 5'GAUCUCG 3' antisense RNA

Genetic constructs designed for gene silencing may also include an inverted repeat. An 'inverted repeat' is a sequence that is repeated where the second half of the repeat is in the complementary strand, e.g.,

5'-GATCTA TAGATC-3' 3'-CTAGAT ATCTAG-5'

The transcript formed may undergo complementary base pairing to form a hairpin structure. Usually a spacer of at least 3-5 bp between the repeated region is required to allow hairpin formation.

Another silencing approach involves the use of a small antisense RNA targeted to the transcript equivalent to an miRNA (Llave et ah, 2002, Science 297, 2053). Use of such small antisense RNA corresponding to polynucleotide of the invention is expressly contemplated. The term genetic construct as used herein also includes small antisense RNAs and other such polynucleotides useful for effecting gene silencing.

Transformation with an expression construct, as herein defined, may also result in gene silencing through a process known as sense suppression (e.g. Napoli et al., 1990, Plant Cell 2, 279; de Carvalho Niebel et al., 1995, Plant Cell, 7, 347). In some cases sense suppression may involve over-expression of the whole or a partial coding sequence but may also involve expression of non-coding region of the gene, such as an intron or a 5' or 3' untranslated region (UTR). Chimeric partial sense constructs can be used to coordinately silence multiple genes (Abbott et al, 2002, Plant Physiol. 128(3): 844-53; Jones et al, 1998, Planta 204: 499-505). The use of such sense suppression strategies to silence the expression of a polynucleotide of the invention is also contemplated.

The polynucleotide inserts in genetic constructs designed for gene silencing may correspond to coding sequence and/or non-coding sequence, such as promoter and/or intron and/or 5' or 3' UTR sequence, or the corresponding gene.

Other gene silencing strategies include dominant negative approaches and the use of ribozyme constructs (Mclntyre, 1996, Transgenic Res, 5, 257)

Pre-transcriptional silencing may be brought about through mutation of the gene itself or its regulatory elements. Such mutations may include point mutations, frameshifts, insertions, deletions and substitutions.

The following are representative publications disclosing genetic transformation protocols that can be used to genetically transform the following plant species: Rice (Alam et al., 1999, Plant Cell Rep. 18, 572); maize (US Patent Serial Nos. 5, 177, 010 and 5, 981, 840); wheat (Ortiz et al, 1996, Plant Cell Rep. 15, 1996, 877); tomato (US Patent Serial No. 5, 159, 135); potato (Kumar et al, 1996 Plant J. 9, : 821); cassava (Li et al, 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et al, 1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al, 1985, Science 227, 1229); cotton (US Patent Serial Nos. 5, 846, 797 and 5, 004, 863); grasses (US Patent Nos. 5, 187, 073, 6. 020, 539); peppermint (Niu et al, 1998, Plant Cell Rep. 17, 165); citrus plants (Pena et al, 1995, Plant Sci.104, 183); caraway (Krens et al, 1997, Plant Cell Rep, 17, 39); banana (US Patent Serial No. 5, 792, 935); soybean (US Patent Nos. 5, 416, 011 ; 5, 569, 834 ; 5, 824, 877 ; 5, 563, 04455 and 5, 968, 830); pineapple (US Patent Serial No. 5, 952, 543); poplar (US Patent No. 4, 795, 855); monocots in general (US Patent Nos. 5, 591, 616 and 6, 037, 522); brassica (US Patent Nos. 5, 188, 958 ; 5, 463, 174 and 5, 750, 871); cereals (US Patent No. 6, 074, 877); gymnosperm tree species and Pine species (Henderson, A. R. and C. Walter, (2006) Genetic Engineering in Conifer Plantation Forestry, Silvae Genetica 55 (6); p253-262). Other species are contemplated and suitable methods and protocols are available in the scientific literature for use by those skilled in the art.

Several further methods known in the art may be employed to alter expression of a nucleotide and/or polypeptide of the invention. Such methods include but are not limited to Tilling (Till et al, 2003, Methods MoI Biol, 2%, 205), so called "Deletagene" technology (Li et al, 2001, Plant Journal 27(3), 235) and the use of artificial transcription factors such as synthetic zinc finger transcription factors, (e.g. Jouvenot et al, 2003, Gene Therapy 10, 513). Additionally antibodies or fragments thereof, targeted to a particular polypeptide may also be expressed in plants to modulate the activity of that polypeptide (Jobling et al, 2003, Nat. Biotechnol., 21(1), 35). Transposon tagging approaches may also be applied. Additionally peptides interacting with a polypeptide of the invention may be identified through technologies such as phase-display (Dyax Corporation). Such interacting peptides may be expressed in or applied to a plant to affect activity of a polypeptide of the invention. Use of each of the above approaches in alteration of expression of a nucleotide and/or polypeptide of the invention is specifically contemplated.

Methods for selecting plants

Methods are also provided for selecting plants altered in at least one of flavonoid production, lignin content, lignin composition, monolignol composition and interunit linkage distribution in developed tracheary elements. Such methods involve testing of plants for altered for the expression of a polynucleotide or polypeptide of the invention. Such methods may be applied at a young age or early developmental stage when the alteration of at least one of flavonoid production, lignin content, lignin composition, monolignol composition and interunit linkage distribution in developed tracheary elements may not necessarily be visible, to accelerate breeding programs. The expression of a polynucleotide, such as a messenger RNA, is often used as an indicator of expression of a corresponding polypeptide. Exemplary methods for measuring the expression of a polynucleotide include but are not limited to Northern analysis, RT-PCR and dot-blot analysis (Sambrook et al. , Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987). Polynucleotides or portions of the polynucleotides of the invention are thus useful as probes or primers, as herein defined, in methods for the identification of plants with altered BIOMASS. The polypeptides of the invention may be used as probes in hybridization experiments, or as primers in PCR based experiments, designed to identify such plants.

Alternatively antibodies may be raised against polypeptides of the invention. Methods for raising and using antibodies are standard in the art (see for example: Antibodies, A Laboratory Manual, Harlow A Lane, Eds, Cold Spring Harbour Laboratory, 1998). Such antibodies may be used in methods to detect altered expression of polypeptides which modulate BIOMASS in plants. Such methods may include ELISA (Kemeny, 1991, A Practical Guide to ELISA, NY Pergamon Press) and Western analysis (Towbin & Gordon, 1994, J Immunol Methods, 72, 313).

These approaches for analysis of polynucleotide or polypeptide expression and the selection of plants with altered expression are useful in conventional breeding programs designed to produce varieties altered in at least one of flavonoid production, lignin content, lignin composition, monolignol composition and interunit linkage distribution in developed tracheary elements.

Plants

The plants of the invention may be grown and either self-ed or crossed with a different plant strain and the resulting hybrids, with the desired phenotypic characteristics, may be identified. Two or more generations may be grown to ensure that the subject phenotypic characteristics are stably maintained and inherited. Plants resulting from such standard breeding approaches also form an aspect of the present invention.

This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows alignment of the amino acid sequences of 34 dehydrin alleles from three pine species. Amino acids in alleles PPH2-PTH11 that are identical to those the top sequence (PPHl) are indicated by dots, the letters indicate amino acid changes, and alignment gaps are indicated by dashes. The translated protein sequence contained an eight-amino-acid, serine-repeat motif and three repeated K-like segments (KIKEK(I/L)PGH) and thus could be classified as acidic SK₃ -type dehydrins (Campbell and Close 1997). The serine (S) polymorphism at amino acid position 191 and the leucine (L) polymorphism at amino acid position 212 are highlighted in grey. PP = Pinus pinaster, PR = Pinus radiata, PT = Pinus taeda. The numbers indicate different alleles.

Figure 2 shows an alignment of the polynucleotide coding sequence of the same dehydrin alleles aligned in Figure 1. The T polymorphism at nucleotide position 571 (that encodes a serine (S) amino acid), and the C polymorphism at nucleotide position 634 (that encodes a leucine (L) amino acid (as shown in Figure I)) are highlighted with grey shading. An 'A' polymorphism at nucleotide positions +462 and +510, which are in strong LD (i.e. R² = 1.0; D = 2.4: D' = 1.0) with the T polymorphism at nucleotide position 571 are underlined and highlighted with grey shading. PP = Pinus pinaster, PR = Pinus radiata, PT = Pinus taeda. The numbers indicate different alleles.

Figure 3 shows the associations between PrDhnl and wood density. The graph shows the average wood densities of trees with nil, one, or two copies of the S/L allele in the CA and GF7 population. The S/L allele refers to a serine (S) polymorphism at amino acid position 191 and a leucine (L) polymorphism at amino acid position 212. EXAMPLES

The invention will now be illustrated with reference to the following non-limiting examples.

Example 1: Demonstration of linkage of a heterozygous dehydrin genotype to wood density in trees

Summary

P. radiata D. Don is grown commercially for wood and pulp throughout NZ, Australia, and Chile . Association tests showed linkage between alleles of the dehydrin gene and wood density in P. radiata. Trees with one copy of an intermediate frequency haplotype had significantly higher wood densities than either homozygote in two New Zealand populations of 226 and 1517 trees respectively.

Materials and methods

Plant material

Two NZ P. radiata populations were assayed in this study and consisted of early (GF7) and later (CA) selections of trees used for breeding. The GF7 (growth and form 7) population of 1517 trees was grown from seed collected from unimproved plantation forests throughout NZ during the 1960s. Parent trees were selected with good growth and form and climbed at age 16-20 years to collect open-pollinated seeds, which were then mixed in drums and planted out. The CA (clonal archive) population consisted of 226 trees used in the NZ radiata breeding program. These trees had been selected based on the superior performance of their progeny in a number of trials, and most were selected for desirable growth and form characteristics (Kumar and Richardson 2005). Over 60% of the CA population consisted of second or third generation offspring from forward selections. Genomic DNA was extracted from needle tissue using a BIO 101 FastDNA (H) kit and a FastPrep FP 120 machine (Savant) following the manufacturer's instructions. Analysis of polymorphisms at the dehydrin locus

In the coding region, five SNPs and two indels were assayed. A single multiplexed-PCR was performed which amplified four SNPs (at bp positions +575, +616, +617, and +705) and two indels (at bp positions +594 and +675) in the coding region. The bp position numbers given here include a 113-bp intron at +268 and a 3-bp indel at +594, so differ from bp position numbers given elsewhere in the document by either +113 or +116 bp. PCR amplifications were carried out under standard conditions. Each SNP was assayed by a different forward primer and the following eight PCR primer pairs were used: PrDhnl+575Fa, PrDhnl+575Fc, PrDhnl+616Ft, PrDhnl+617Fgc, PrDhnl+617Fgt, PrDhnl+705Fc, PrDhnl+705Ft, and PrDhnlR (Table 1). PCR products were diluted 100-fold in distilled water, and 1 μl of diluted PCR product was added to 0.01 μl of GS LIZ 500 size standard (Applied Biosystems), and 9.9 μl Hi-Di formamide (Applied Biosystems). PCR products were electrophoresed through POP4 polymer (Applied Biosystems) in a 36-cm capillary array on a 3100 DNA analyzer using standard electrophoretic conditions (Applied Biosystems). The electrophoresis data was analysed using GENESCAN ANALYSIS v3.7 and GENOTYPER v3.7 software (Applied Biosystems).

Table 1 PCR primer sequences

Position in Name Sequence (5' to 3') PrDhnl (bp)

PrDhn 1 F CGAAGGAC AAGAGCA AAGAAAAAC (SEQ ID NO:71 ) +442 to +465

PrDhn 1 R TCTTCCTCTTCCGC ATCATT (SEQ ID NO:72) +814 to +795

PrDhnl+575Fa 6FAM-CGGGACACCAGGAAAAACTA (SEQ ID NO:73) +556 to +575

PrDhnl+575Fc HEX-CGGGACACCAGGAAAAACTC (SEQ ID NO:74) +556 to +575

PrDhn 1+616Ft HEX-CATTCTTCAGATGAGTGTGAGGT (SEQ ID NO:75) +593 to +616

PrDhn 1 +617Fgc H EX-TTCTTC AG ATG AGTGTGG AGGC (SEQ ID NO:76) +596 to +617

PrDhnl+617Fgt 6FAM-TTCTTCAGATGAGTGTGGAGGT (SEQ ID NO:77) +596 to +617

PrDhnl+705Fc HEX-CCCTGGTGATGGAAAGTACC (SEQ ID NO:78) +686 to +705

PrDhnl+705Ft 6FAM-CTCTGGTGATGGAAAGCACT (SEQ ID NO:79) +686 to +705

PrDhnl+750Fc 6FAM-AGGAGAAGAAGTTGGGT ATGC (SEQ ID NO:80) +730 to +750

PrDhn 1+750Fg HEX-AGGAGAAGAAGTTGGGTATGG (SEQ ID NO:81) +730 to +750

PrDhn 1-463Fa VIC-GCGTAGTAAAACATATTGACCTAACTA (SEQ ID NO:82) -437 to -463

6F AM-GCGTAGTAAAACAT ATTGACCT AACTG (SEQ ID

PrDhnl-463Fg NO:83) -437 to -463 A SNP at position +750 bp was assayed separately using PCR primers: PrDhnl+750Fc, PrDhnl+750Fg, and PrDhnlR (Table 1) (i.e. each SNP was amplified with a different primer). Likewise, in the promoter region, one SNP (at -463 bp) and three indels (at -504, -634, and -692 bp) were amplified using PCR primers: PrDhnl-463Fa, PrDhnl-463Fg, and PrDhnlpromR (Table 1). All products were analysed on the 3100 DNA analyzer as described in the proceeding paragraph.

The SNP specific primers PrDhnl+750Fc and PrDhnl+750Fg assay the leucine (L) or valine (V) polymorphism respectively at amino acid position 212. The serine (S) polymorphism at amino acid position 191 was assayed using the SNP specific primer PrDhnl+575Fa. The A nucleotide at bp position +575 (or bp position +462 (excluding the 113 bp intron)) is in strong LD (i.e. R = 1.0; D = 2.4: D' = 1.0) with the T nucleotide polymorphism that encodes the serine (S) at amino acid position 191. Likewise the proline (P) polymorphism at amino acid position 191 was assayed using the SNP specific primer PrDhnl+575Fc, as the C nucleotide at bp position +575 (or bp position +462 (excluding the 1 13 bp intron)) is in strong LD (i.e. R² = 1.0; D = 2.4: D' = 1.0) with the C nucleotide polymorphism that encodes the proline (P) at amino acid position 191.

Wood property measurements

In the GF7 population, wood density and ring widths were measured from 5-mm cores (spanning the pith to the bark) by x-ray densitometry (Cown and Clement 1983). Each core was collected at breast height (1.4 m). For each individual, wood densities were averaged across rings 1-10, and were standardized by site and silvicultural treatment. The sum of the ring widths from rings five to twelve were calculated for each core and used as a measure of growth rate at breast height. A mixed model with fixed effects for experiment and treatment and random plot effects was fitted to the ring width data. The standardised ring widths were calculated as the standardized residuals of this model. Calculations were performed using the R language for statistical graphics available at http://www.r-proiect.org/ (Ihaka and Gentleman 1996). In the CA population, breeding values for both wood density (Kumar and Richardson 2005) and diameter at breast height (unpublished data) were calculated for each tree based on the performance of their offspring. Association Tests

Tests for an association between gene polymorphisms (individual SNPs or haplotypes), average wood density and ring width were performed in PO WERMARKER V3.25 which is available at http://statgen.ncsu.edu/powermarker/ using a single-locus F-test (Liu and Muse 2005).

Genetic mapping

The PrDhnl locus (from to +442 to +814 bp (within exon-2)) was amplified using PCR primers PrDhnl F and PrDhnl R (Table 1), in the parents and progeny of a full-sib mapping pedigree (i.e. tree 268.405 crossed with tree 268.345 (both trees are present in the NZ CA population)). PCR products were either electrophoresed on 6% non-denaturing gels and stained with ethidium bromide, or electrophoresed in an ABI 377. Length polymorphisms and heteroduplexes were scored for each individual, and the locus was placed onto existing parent-specific genetic linkage maps (Wilcox, unpublished data for the 268.405 x 268.345 pedigree) using a pseudo-testcross strategy (Grattapaglia et al. 1995) using MAPMAKER Macintosh V2.0 (Lander et al. 1987). The criteria for accepting linkage between loci were LOD > 5, θ< 0. 2. After placing the locus into its most likely linkage groups, map locations were determined using the 'TRY' command. Wood density was measured from 5-mm cores in all trees using the maximum moisture content method (Smith 1954). Statistical associations between the PrDhnl locus and wood density (measured on 4 year-old trees) were analysed in 400 selectively genotyped trees and 998 randomly selected trees using single marker ANOVA (as implemented in SAS V8.0), followed by single marker regression and interval mapping as implemented in Qgene V 3.06 (Nelson 1997). The 400 selectively genotyped trees were chosen from the high and low tails of two independent 1500- tree populations, and 998 trees were chosen at random from one of the 1500-tree populations. Chromosome-wise Type 1 error rates of 5% were determined using 10,000 permutations, corresponding to a minimum LOD threshold of 2.7. Results

Molecular basis of polymorphisms

Association tests in two NZ populations of P. radiata

In order to test whether polymorphisms, or haplotypes thereof, at the dehydrin locus were associated with wood density trait variation in the GF7 or CA population, association tests were performed between individual tree phenotypes and the PrDhnl haplotypes. Trees that contained a single S/L allele (i.e. were heterozygous for S/L) had significantly higher wood densities (an average increase of 3.7 to 5.8 kg m^"3, or an increase of 0.1-0.2 standard deviations from the mean), than trees that contained either no or two S/L alleles respectively (Figure 3). This association was detected in both the GF7 and CA populations (p-value = 0.008 and 0.056 respectively), with a significant over-dominant effect (p-value < 0.003) in both.

PrDHNl maps to a QTL for wood density in a P. radiata pedigree

A single second-generation pedigree from the CA population was chosen for inheritance analysis, based on it being the largest known full-sib planting in NZ at a single site. The pedigree was tested for an association between wood density and alleles of the PrDhnl gene, in which it was fully informative. PrDhnl mapped to a locus that was associated with wood density in a sample of over 400 selectively genotyped trees and an independent sample of 998 randomly selected trees (p-value < 0.005). The PrDhnl gene also mapped to a QTL for wood density in a P. pinaster mapping pedigree (data not shown).

Using the estimation methods described by Darvasi and Soller (1997), a 95% confidence interval around the quantitative trait locus (QTL) in the P radiata pedigree was approximately 12.6 cM. These results confirm the associations of Dhnl and wood density found in the general population. Discussion

Evidence has been presented for a heterozygous genetic effect at the PrDhnl locus in the GF7 and CA population of P. radiata, where trees heterozygous for a S/L allele showed an increased wood density.

The results demonstrated that genetic variation at the dehydrin locus underpins changes in wood density, a crucial trait in timber tree breeding. This study provides the first evidence that variation at a single gene can affect wood density.

References

Campbell, S. A. and T. J. Close (1997). Dehydrins: genes, proteins, and associations with phenotypic traits. New Phytol. 137: 61-74.

Cown, D. J. and B. C. Clement (1983). A wood densitometer using direct scanning with X-rays. Wood ScL Technol. 17: 91-99.

Darvasi, A. and M. Soller (1997). A simple method to calculate resolving power and confidence interval of QTL map location. Behav. Genet. 27: 125-132.

Grattapaglia, D. G., F. L. Bertolucci and R. R. Sederoff (1995). Genetic mapping of QTLs controlling vegetative propagation in Eucalyptus grandis and E europhylla using a pseudo- testcross strategy and RAPD markers. Theor. Appl. Genet. 90: 933-947.

Ihaka, R. and R. Gentleman (1996). R: a language for data analysis and graphics. J. Comput. Graphical Stat. 5: 299-314.

Kumar, S. and T. E. Richardson (2005). Inferring relatedness and heritability using molecular markers in radiata pine. MoI. Breed. 15: 55-64. Lander, E., P. Green, J. Abrahamson, A. Barlow, M. Daly, S. Lincoln and L. Newburg (1987). MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1: 174-181.

Liu, K. and S. V. Muse (2005). PowerMarker: an integrated analysis environment for genetic marker analysis. Bioinformatics 21: 2128-2129.

Nelson, J. C. (1997). QGENE: software for marker-based genomic analysis and breeding. MoI. Breed. 3: 239-245.

Smith, D. M. (1954). Maximum moisture content method for determining specific gravity of small wood samples. Forest Products Laboratory Report No. 2014. Wisconsin, US Forest Service.

The above Examples illustrate practice of the invention. It will be appreciated by those skilled in the art that numerous variations and modifications may be made without departing from the spirit and scope of the invention.

Summary of Sequences

N/A = not applicable

Claims

CLAIMS:

1. A method for identifying a tree with a genotype indicative of at least one of increased wood density and increased cell wall thickness, the method including detecting in the tree, or a sample derived from the tree, by direct or indirect methods, the presence of:

(i) a first allele of the dehydrin gene that encodes a dehydrin protein including a serine

(S) at amino acid position 191 and a leucine (L) at amino acid position 212; and (ii) a second allele of the dehydrin gene that encodes a dehydrin protein including at least one of (a) a proline (P) at amino acid position 191, or

(b) a valine (V) at amino acid position 212.

2. The method of claim 1, wherein the dehydrin protein of the first allele, comprises a sequence with at least 70% identity to the polypeptide sequence of any one of SEQ ID NO: 13, 16, 17 and 20.

3. The method of claim 1 or 2, wherein the dehydrin protein of the first allele has the sequence of any one of SEQ ID NO: 13, 16, 17 and 20.

4. The method of any preceding claim wherein the first dehydrin allele comprises a sequence with at least 70% identity to the polynucleotide sequence of any one of SEQ ID NO: 47, 50, 51 and 54.

5. The method of any preceding claim wherein the first dehydrin allele contains a codon at nucleotide positions 571-573 encoding the serine (S) at amino acid position 191 in the dehydrin protein.

6. The method of claim 5, wherein the codon is TCT.

7. The method of any preceding claim wherein the first dehydrin allele contains a codon at nucleotide positions 634-636 encoding the leucine (L) at amino acid position 212 in the dehydrin protein.

8. The method of claim 7, wherein the codon is CTT.

9. The method of any preceding claim wherein the first dehydrin allele comprises the polynucleotide sequence of any one of SEQ ID NO: 47, 50, 51 and 54.

10. The method of any preceding claim wherein the dehydrin protein of the second allele comprises proline (P) at amino acid position 191.

1 1. The method of any preceding claim wherein the dehydrin protein of the second allele comprises a sequence with at least 70% identity to the polypeptide sequence of any one of SEQ

ID NO: 1-12 and 22-34.

12. The method of any preceding claim wherein the dehydrin protein of the second allele comprises the polypeptide sequence of any one of SEQ ID NO: 1-12 and 22-34.

13. The method of any preceding claim wherein second dehydrin allele includes a codon encoding a proline (P) residue at amino acid position 191.

14. The method of any preceding claim wherein second dehydrin allele comprises a sequence with at least 70% identity to the polynucleotide sequence of any one of SEQ ID NO: 35-46 and

56-68.

15. The method of any preceding claim wherein second dehydrin allele contains a codon at nucleotide positions 571-573 encoding the proline (P) at amino acid position 191.

16. The method of claim 15, wherein the codon is CCT.

17. The method of any preceding claim wherein second dehydrin allele comprises the polynucleotide sequence of any one of SEQ ID NO: 35-46 and 56-68.

18. The method of any preceding claim wherein the dehydrin protein of the second allele comprises valine (V) at amino acid position 212.

19. The method of any preceding claim wherein the dehydrin protein of the second allele comprises a sequence with at least 70% identity to the polypeptide sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.

20. The method of any preceding claim wherein the dehydrin protein of the second allele comprises the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.

21. The method of any preceding claim wherein the second dehydrin allele includes a codon encoding a valine (V) at amino acid position 212.

22. The method of any preceding claim wherein the second dehydrin allele comprises a sequence with at least 70% identity to the polynucleotide sequence of any one of SEQ ID NO: 48, 49, 52, 53 and 55.

23. The method of any preceding claim wherein second dehydrin allele contains a codon at nucleotide positions 634-636 encoding the valine (V) at amino acid position 212.

24. The method of claim 23 wherein the codon is GTT.

25. The method of any preceding claim wherein the second dehydrin allele comprises the polynucleotide sequence of any one of SEQ ID NO: 48, 49, 52, 53 and 55.

26. The method of any preceding claim wherein the presence of first and second alleles is in LD with the increased wood density and/or cell wall thickness trait.

27. The method of any preceding claim wherein the presence of first and/or second alleles is detected indirectly by detecting a marker that is linked to the first and/or second allele.

28. The method of claim 27 wherein the marker is in linkage disequilibrium (LD) with the first or second allele.

29. The method of any one of claims 1 to 26 wherein the allele is directed directly by detecting the presence of nucleotides, or encoded amino acids, distinctive of the allele.

30. The method of any preceding claim wherein presence of the allele or nucleotides is detected via a polymerase chain reaction (PCR) step.

31. The method of claim 30 wherein a PCR product comprising the nucleotide of the marker is amplified using primers comprising sequence complimentary to sequence of the genome flanking the nucleotide or marker.

32. The method of claim 31 wherein at least one primer capable of hybridising to the sequence of the genome under standard PCR conditions is used.

33. The method of claim 32 wherein the primer comprises at least 10 contiguous nucleotides of the sequence of any one of SEQ ID NO: 35 to 68, or complements thereof.

34. A method for selecting a tree with a genotype indicative of increased wood density and/or cell wall thickness, the method comprising selecting a tree identified by the method of any one of claims 1 to 33.

35. An isolated polynucleotide encoding a polypeptide with the sequence of any one of SEQ ID NO: 1-12 and 22-34 or a variant thereof, wherein the variant is a polypeptide capable of increasing at least one of wood density and cell wall thickness, when expressed in a tree.

36. The isolated polynucleotide of claim 35, wherein the polypeptide has at least 70% identity to the sequence of any one of SEQ ID NO: 1-12 and 22-34.

37. The isolated polynucleotide of claim 35 or 36, wherein the polypeptide includes a proline (P) residue at amino acid position 191.

38. The isolated polynucleotide of claim 35 or 36, wherein the polypeptide comprises the sequence of any one of SEQ ID NO: 1-12 and 22-34.

39. An isolated polynucleotide comprising the sequence of any one of SEQ ID NO: 35-46 and 56-68 or a variant thereof, wherein the variant encodes a polypeptide capable of increasing at least one of wood density and cell wall thickness, when expressed in a tree.

40. The isolated polynucleotide of claim 39 comprising a sequence with at least 70% identity to the polynucleotide sequence of any one of SEQ ID NO: 35-46 and 56-68.

41. The isolated polynucleotide of claim 39 or 40 including a codon encoding proline (P) at nucleotide positions 571-573.

42. The isolated polynucleotide of any one of claims 39 to 41 comprising the sequence of any one of SEQ ID NO: 35-46 and 56-68.

43. An isolated polypeptide with the sequence of any one of SEQ ID NO: 1-12 and 22-34 or a variant thereof, wherein the variant is a polypeptide capable of increasing at least one of wood density and cell wall thickness, when expressed in a tree.

44. An isolated polynucleotide encoding a polypeptide with the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21 or a variant thereof, wherein the variant is a polypeptide capable of increasing at least one of wood density and cell wall thickness, when expressed in a tree.

45. The isolated polynucleotide of claim 44 wherein the polypeptide has at least 70% identity to the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.

46. The isolated polynucleotide of claim 44 or 45 wherein the polypeptide includes a valine (V) residue at amino acid position 212.

47. The isolated polynucleotide of any one of claims 44 to 46 wherein the polypeptide comprises the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.

48. An isolated polynucleotide comprising the sequence of any one of SEQ ID NO: 48, 49, 52, 53 and 55 or a variant thereof, wherein the variant encodes a polypeptide capable of increasing at least one of wood density and cell wall thickness, when expressed in a tree.

49. The polynucleotide of claim 48 comprising a sequence with at least 70% identity to the polynucleotide sequence of any one of SEQ ID NO: 48, 49, 52, 53 and 55.

50. The isolated polynucleotide of claim 48 or 49 including a codon encoding valine (V) at nucleotide positions 634-636.

51. The isolated polynucleotide of any one of claims 48 to 50 comprising the sequence of any one of SEQ ID NO: 48, 49, 52, 53 and 55.

52. An isolated polypeptide with the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21 or a variant thereof, wherein the variant is a polypeptide capable of increasing at least one of wood density and cell wall thickness, when expressed in a tree.

53. A genetic construct comprising a polynucleotide of any one of claims 35 to 42 and 44 to 51.

54. The genetic construct of claim 53, that is an expression construct.

55. A host cell comprising a polynucleotide of any one of claims 35 to 42 and 44 to 51, or a genetic construct of claim 53 or 55.

56. The host cell of claim 55 genetically modified to express a polynucleotide of any one of claims 35 to 42 and 44 to 51, or a polypeptide of claim 43 or 52.

57. The host cell of claim 55 or 56 that is a plant cell.

58. A method of producing a plant with at least one of increased wood density and increased cell wall thickness, the method comprising transformation of a plant with: a) a polynucleotide of any one of claims 35 to 42 and 44 to 51 ; b) a polynucleotide comprising a fragment, of at least 15 nucleotides in length, of the polynucleotide of a); or c) a polynucleotide comprising a complement of the polynucleotide of a) or b).

59. The method of claim 58, wherein the plant is transformed with a genetic construct or vector comprising the polynucleotide.

60. The method of claim 60 or 61 in which the plant produced has been transformed to comprise: (i) a first allele of the dehydrin gene that encodes a dehydrin protein including a serine

(S) at amino acid position 191 and a leucine (L) at amino acid position 212; and (ii) a second allele of the dehydrin gene that encodes a dehydrin protein including at least one of (a) a proline (P) at amino acid position 191, or (b) a valine (V) at amino acid position 212.

61. A plant which: a) comprises a plant cell of claim 57, or b) is produced by the method of any one of claims 58 to 60

62. The plant of claim 61, wherein the plant is a tree and the plant cell is a tree cell.

63. A part, fruit, seed, harvested material, propagule or progeny of a plant of claim 61 or 62.

64. A part, fruit, seed, harvested material, propagule or progeny of claim 63, that is genetically modified to comprise at least one polynucleotide of any one of claims 35 to 42 and 44 to 51 or a genetic construct of construct of claim 53 or 54.

65. A group of trees selected by the method of claim 34.