WO2010002277A1 - Procédés et compositions pour améliorer des arbres - Google Patents

Procédés et compositions pour améliorer des arbres Download PDF

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Publication number
WO2010002277A1
WO2010002277A1 PCT/NZ2009/000128 NZ2009000128W WO2010002277A1 WO 2010002277 A1 WO2010002277 A1 WO 2010002277A1 NZ 2009000128 W NZ2009000128 W NZ 2009000128W WO 2010002277 A1 WO2010002277 A1 WO 2010002277A1
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Prior art keywords
sequence
polynucleotide
seq
allele
dehydrin
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PCT/NZ2009/000128
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English (en)
Inventor
Sheree Alice Cato
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New Zealand Forest Research Institute Limited
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Publication of WO2010002277A1 publication Critical patent/WO2010002277A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers

Definitions

  • 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.
  • 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.
  • 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.
  • measurement of wood density and/or cell wall thickness is time consuming and expensive.
  • 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 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.
  • 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.
  • 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:
  • 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.
  • polypeptide of the first dehydrin allele polypeptide has the sequence of any one of SEQ ID NO: 13, 16, 17 and 20.
  • 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.
  • polynucleotide of the first dehydrin allele comprises the polynucleotide sequence of any one of SEQ ID NO: 47, 50, 51 and 54.
  • the polynucleotide contains a codon at nucleotide positions 571-573 encoding the serine (S) at amino acid position 191.
  • the codon is TCT.
  • the polynucleotide contains T at nucleotide position 571.
  • the polynucleotide contains a codon at nucleotide positions 634-636 encoding the leucine (L) at amino acid position 212.
  • the codon is CTT.
  • the polynucleotide contains C at nucleotide position 634.
  • the second dehydrin allele polypeptide comprises proline (P) at amino acid position i91.
  • 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.
  • polypeptide of the first dehydrin allele polypeptide has the sequence of any one of SEQ ID NO: 1-12 and 22-34.
  • the second dehydrin allele polynucleotide includes a codon encoding a proline (P) residue at amino acid position 191.
  • 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.
  • polynucleotide of the second dehydrin allele comprises the polynucleotide sequence of any one of SEQ ID NO: 35-46 and 56-68.
  • the polynucleotide contains a codon at nucleotide positions 571-573 encoding the proline (P) at amino acid position 191.
  • the codon is CCT.
  • the polynucleotide contains C at nucleotide position 571.
  • the second dehydrin allele polypeptide comprises valine (V) at amino acid position 212.
  • 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.
  • polypeptide of the second dehydrin allele polypeptide has the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.
  • the second dehydrin allele polynucleotide includes a codon encoding a valine (V) at amino acid position 212.
  • 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.
  • polynucleotide of the second dehydrin allele comprises the polynucleotide sequence of any one of SEQ ID NO: 48, 49, 52, 53 and 55.
  • the polynucleotide contains a codon at nucleotide positions 634-636 encoding the valine (V) at amino acid position 212.
  • the codon is GTT.
  • the polynucleotide contains G at nucleotide position 634.
  • the presence of the pair of heterozygous alleles is in LD with the increased wood density and/or cell wall thickness trait.
  • 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 R 2 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.
  • the marker is in linkage disequilibrium (LD) with the first or second allele.
  • 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.
  • the marker is in LD with the allele at a R 2 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.
  • the protein isoforms are encoded by corresponding nucleic acid alleles.
  • 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.
  • the alleles or markers are detected using a polymerase chain reaction (PCR) step.
  • PCR polymerase chain reaction
  • 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.
  • the PCR is performed using at least one primer selected from those set forth in Table 2
  • the PCR is performed using at least one primer pair selected from those set forth in Table 2
  • probe-based 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.
  • 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.
  • 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.
  • 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.
  • the tree cell or tree is transformed to express an S/L dehydrin allele polypeptide.
  • 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.
  • 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.
  • 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.
  • 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.
  • the dehydrin protein comprises the sequence of any one of SEQ ID NO: 13, 16, 17 and 20.
  • 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.
  • polynucleotide comprises the polynucleotide sequence of any one of SEQ ID NO: 47, 50, 51 or 54.
  • 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.
  • the polynucleotide transformed encodes a dehydrin protein including a proline (P) residue at amino acid position 191.
  • 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.
  • the dehydrin protein comprises the sequence of any one of SEQ ID NO: 1-12 and 22- 23.
  • 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.
  • polynucleotide comprises the polynucleotide sequence of any one of SEQ ID NO: 35-46 and 56-58.
  • 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.
  • S/L serine/leucine
  • the polynucleotide transformed encodes a dehydrin protein including a valine (V) residue at amino acid position 212.
  • 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.
  • the dehydrin protein comprises the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.
  • 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.
  • polynucleotide comprises the polynucleotide sequence of any one of SEQ ID NO: 48, 49, 52, 53 and 55.
  • 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.
  • S/L serine/leucine
  • the invention provides a tree cell or tree produced by a method of the invention.
  • Serine/Leucine (S/L) allele polynucleotides encoding polypeptides
  • 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.
  • polypeptide has at least 70% identity to the sequence of any one of SEQ ID NO: 13, 16, 17 and 20.
  • the polypeptide includes a serine (S) residue at amino acid position 191 and a leucine residue at amino acid position 212.
  • polypeptide comprises the sequence of any one of SEQ ID NO: 13, 16, 17 and 20.
  • polypeptide consists of the sequence of any one of SEQ ID NO: 13, 16, 17 and 20.
  • Serine/Leucine (S/L) allele polynucleotides S/L allele polynucleotides
  • 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.
  • 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.
  • the polynucleotide includes a codon encoding serine (S) at nucleotide positions 571-573 and a codon encoding leucine (L) at nucleotide positions 634-636.
  • S serine
  • L leucine
  • the codon at nucleotide position 571-573 is TCT.
  • the codon at nucleotide position 571-573 is TCT.
  • polynucleotide comprises the sequence of any one of SEQ ID NO: 47, 50, 51 or
  • polynucleotide consists of the sequence of any one of SEQ ID NO: 47, 50, 51 or 54.
  • 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.
  • polypeptide has at least 70% identity to the sequence of any one of SEQ ID NO: 13, 16, 17 and 20.
  • polypeptide includes a serine (S) residue at amino acid position 191 and a leucine (L) residue at amino acid position 212.
  • polypeptide comprises the sequence of any one of SEQ ID NO: 13, 16, 17 and 20.
  • polypeptide consists of the sequence of any one of SEQ ID NO: 13, 16, 17 and 20.
  • Proline (P) allele polynucleotides encoding polypeptides
  • 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.
  • polypeptide has at least 70% identity to the sequence of any one of SEQ ID NO: 1-12 and 22-34.
  • polypeptide includes a proline (P) residue at amino acid position 191.
  • polypeptide comprises the sequence of any one of SEQ ID NO: 1-12 and 22-34.
  • polypeptide consists of the sequence of any one of SEQ ID NO: 1-12 and 22-34.
  • 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.
  • 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.
  • the polynucleotide includes a codon encoding proline (P) at nucleotide positions 571-573.
  • the codon at nucleotide position 571-573 is CCT.
  • the polynucleotide comprises the sequence of any one of SEQ ID NO: 35-46 and 56- 68.
  • polynucleotide consists of the sequence of any one of SEQ ID NO: 35-46 and 56- 68.
  • 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.
  • polypeptide has at least 70% identity to the sequence of any one of SEQ ID NO: 1-12 and 22-34.
  • polypeptide includes a serine (P) residue at amino acid position 191.
  • polypeptide comprises the sequence of any one of SEQ ID NO: 1-12 and 22-34.
  • polypeptide consists of the sequence of any one of SEQ ID NO: 1-12 and 22-34.
  • V Valine (V) allele polynucleotides encoding polypeptides
  • 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.
  • polypeptide has at least 70% identity to the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.
  • the polypeptide includes a valine (V) residue at amino acid position 212.
  • V valine
  • the polypeptide comprises the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.
  • polypeptide consists of the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.
  • V Valine (V) allele polynucleotides
  • 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.
  • 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.
  • the polynucleotide includes a codon encoding valine (V) at nucleotide positions 634-636.
  • V valine
  • the codon at nucleotide position 634-636 is GTT.
  • polynucleotide comprises the sequence of any one of SEQ ID NO: 48, 49, 52, 53 and 55.
  • polynucleotide consists of the sequence of any one of SEQ ID NO: 48, 49, 52, 53 and 55.
  • 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.
  • the polypeptide has at least 70% identity to the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.
  • the polypeptide includes a valine (V) residue at amino acid position 212.
  • polypeptide comprises the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.
  • polypeptide consists of the sequence of any one of SEQ ID NO: 14, 15, 18, 19 and 21.
  • the invention provides a polynucleotide encoding a polypeptide of the invention.
  • the invention provides a genetic construct comprising a polynucleotide of the invention.
  • the genetic construct is an expression construct.
  • the invention provides a vector comprising a polynucleotide, genetic construct or expression construct of the invention.
  • the invention provides a host cell comprising a polynucleotide, genetic construct or expression construct of the invention.
  • the invention provides a host cell genetically modified to express a polynucleotide of the invention.
  • 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.
  • the invention provides a plant which comprises a plant cell of the invention.
  • the plants of the invention are trees, and the plant cells are tree cells.
  • the invention provides a group of trees selected by a method of the invention.
  • the invention provides a tree cell or tree produced by a method of the invention.
  • 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 purgeds, Picea rubens, Picea sitchensis, Pinus banksiana, Pinus brutia, Pinus caribaea, Pinus clausa, Pinus contorta, Pinus coulteri, Pinus e
  • 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 ⁇ gida, 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.
  • Pinus species include Pinus radiata and Pinus taeda.
  • a particularly preferred Pinus species is Pinus radiata.
  • 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 are part of a large group of highly hydrophilic proteins known as LEA (Late).
  • DHNs a conserved, lysine-rich 15- amino acid domain, EKKGIMDKIKEKLPG, named the K-segment. It is usually present near the C-terminus.
  • K-segment a conserved, lysine-rich 15- amino acid domain
  • 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),
  • 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.
  • 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 ⁇ /.,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 ( ⁇ m).
  • 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 ⁇ /.,1983, Wood Sci. Technol. 17, 91) and SilviScan (Evans et al, 1995, Appita J. 48, 134).
  • Polymorphism is a condition in DNA in which the most frequent variant (or allele) has a population frequency which does not exceed 99%.
  • linkage disequilibrium 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 R 2 , 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).
  • 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.”
  • (ATC)4 refers to a tri-nucleotide ATC motif that is repeated four times in a particular sequence.
  • (ATC)4 is a shorthand version of "ATCATCATCATC.”
  • complement of a SSR motif refers to a complementary strand of the represented motif.
  • complement of (ATG) motif is (TAC).
  • 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.
  • 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.
  • 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.
  • polynucleotide(s), 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.
  • 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.
  • 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.
  • polynucleotide(s), 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.
  • 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.
  • 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.
  • polypeptide 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • variants of the inventive polypeptides and polypeptides possess biological activities that are the same or similar to those of the inventive polypeptides or polypeptides.
  • variant with reference to polypeptides and polypeptides encompasses all forms of polypeptides and polypeptides as defined herein.
  • 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
  • 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.
  • the parameter -F F turns off filtering of low complexity sections.
  • the parameter -p selects the appropriate algorithm for the pair of sequences.
  • 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).
  • 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/.
  • GAP Global Sequence Alignment
  • 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.
  • 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 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.
  • variant polynucleotides of the present invention hybridize to a specified polynucleotide sequence, or complements thereof under stringent conditions.
  • stringent conditions 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.
  • Tm melting temperature
  • 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 65 0 C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65 0 C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65 0 C.
  • exemplary stringent hybridization conditions are 5 to 10° C below Tm.
  • Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length) 0 C.
  • 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.
  • 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
  • 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/).
  • BLASTP from the BLAST suite of programs, version 2.2.5 [Nov 2002]
  • bl2seq which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
  • 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/
  • GAP Human, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227- 235.
  • suitable global sequence alignment programs for calculating polypeptide sequence identity.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.,
  • 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.
  • 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.
  • polypeptides of the invention can be isolated by using a variety of techniques known to those of ordinary skill in the art.
  • 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.
  • PCR polymerase chain reaction
  • the polypeptides of the invention can be amplified using primers, as defined herein, derived from the polynucleotide sequences of the invention.
  • hybridization probes include use of all, or portions of, the polynucleotides set forth herein as hybridization probes.
  • 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.
  • 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.
  • 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.
  • standard molecular biology approaches can be utilized (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).
  • transgenic plant from a particular species, 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.
  • 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.
  • Variant polynucleotides may be identified using PCR-based methods (Mullis et al, Eds. 1994 The Polymerase Chain Reaction, Birkhauser).
  • 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.
  • 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.
  • variant sequences of the invention 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.
  • NCBI National Center for Biotechnology Information
  • 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.
  • BLAST family of algorithms including BLASTN, BLASTP, and BLASTX
  • BLASTN, BLASTP, and BLASTX 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.
  • the probability of finding a match by chance in that database is 1% or less using the BLASTN, BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.
  • Pattern recognition software applications are available for finding motifs or signature sequences.
  • MEME Multiple Em for Motif Elicitation
  • MAST Motif Alignment and Search Tool
  • 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 Boiroch 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.
  • PROSITE database www.expasy.org/prosite
  • the PROSITE database 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.
  • polypeptides of the invention 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.
  • 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.
  • 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
  • polypeptides and variant polypeptides of the invention may be expressed recombinantly in suitable host cells and separated from the cells as discussed below.
  • 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).
  • 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.
  • 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.
  • strategies for genetically manipulating plants are available (e.g. Birch, 1997, Ann Rev Plant Phys Plant MoI Biol, 48, 297).
  • 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.
  • CaMV cauliflower mosaic virus
  • Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators the Zea mays zin gene terminator
  • the Oryza sativa ADP-glucose pyrophosphorylase terminator the Solarium tuberosum PI-II terminator.
  • NPT II neomycin phophotransferase II gene
  • aadA gene which confers spectinomycin and streptomycin resistance
  • phosphinothricin acetyl transferase bar gene
  • Ignite AgrEvo
  • Basta Hoechst
  • hpt hygromycin phosphotransferase gene
  • 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
  • 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.,
  • 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.,
  • the transcript formed may undergo complementary base pairing to form a hairpin structure.
  • 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).
  • 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.
  • 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.
  • nucleotide and/or polypeptide of the invention 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.
  • 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 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.
  • 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).
  • 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.
  • 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 SK 3 -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
  • the C polymorphism at nucleotide position 634 that encodes a leucine (L) amino acid (as shown in Figure I)
  • PP Pinus pinaster
  • PR Pinus radiata
  • PT Pinus taeda.
  • the numbers indicate different alleles.
  • FIG. 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.
  • Example 1 Demonstration of linkage of a heterozygous dehydrin genotype to wood density in trees
  • 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.
  • 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).
  • 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
  • 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 ⁇ l of diluted PCR product was added to 0.01 ⁇ l of GS LIZ 500 size standard (Applied Biosystems), and 9.9 ⁇ l 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).
  • PrDhn 1 R TCTTCCTCTTCCGC ATCATT (SEQ ID NO:72) +814 to +795
  • 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).
  • PCR primers PrDhnl+750Fc, PrDhnl+750Fg, and PrDhnlR (Table 1) (i.e. each SNP was amplified with a different primer).
  • PCR primers at -463 bp
  • three indels at -504, -634, and -692 bp
  • 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.
  • 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, ⁇ 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).
  • 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).
  • PowerMarker an integrated analysis environment for genetic marker analysis. Bioinformatics 21: 2128-2129.

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Abstract

La présente invention concerne un procédé pour identifier un arbre ayant un génotype indicatif d’au moins l’un parmi une densité de bois augmentée et une épaisseur de paroi cellulaire augmentée, le procédé comprenant la détection dans l’arbre, ou un échantillon dérivé de l’arbre, par des procédés directs ou indirects, de la présence de : (i) un premier allèle du gène de la déhydrine qui code pour une protéine de déhydrine comprenant une sérine (S) à la position d’acide aminé (191) et une leucine (L) à la position d’acide aminé (212); et (ii) un deuxième allèle du gène de la déhydrine qui code pour une protéine de déhydrine comprenant au moins l’un de (a) une proline (P) à la position d’acide aminé (191), ou (b) une valine (V) à la position d’acide aminé (212). L’invention concerne en outre les polynucléotides isolés de tels allèles, des constructions, des cellules hôtes, des cellules de plante et des plantes comprenant de tels polynucléotides. L’invention concerne en outre des procédés pour produire des plantes ayant au moins l’un parmi une densité de bois augmentée et une épaisseur de paroi cellulaire augmentée, utilisant les polynucléotides de l’invention, et des plantes produites par les procédés.
PCT/NZ2009/000128 2008-06-30 2009-06-30 Procédés et compositions pour améliorer des arbres WO2010002277A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050108791A1 (en) * 2001-12-04 2005-05-19 Edgerton Michael D. Transgenic plants with improved phenotypes
WO2006085769A2 (fr) * 2005-02-09 2006-08-17 Nsure Holding, B.V. Procedes et moyens permettant de determiner et de conferer la tolerance au stress chez les plantes
US20080148432A1 (en) * 2005-12-21 2008-06-19 Mark Scott Abad Transgenic plants with enhanced agronomic traits

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050108791A1 (en) * 2001-12-04 2005-05-19 Edgerton Michael D. Transgenic plants with improved phenotypes
WO2006085769A2 (fr) * 2005-02-09 2006-08-17 Nsure Holding, B.V. Procedes et moyens permettant de determiner et de conferer la tolerance au stress chez les plantes
US20080148432A1 (en) * 2005-12-21 2008-06-19 Mark Scott Abad Transgenic plants with enhanced agronomic traits

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Title
ALTMAN, A.: "From Plant Tissue Culture to Biotechnology: Scientific Revolutions, Abiotic Stress Tolerance, and Forestry.", VITRO CELLULAR AND DEVELOPMENTAL BIOLOGY. - PLANT, vol. 39, March 2003 (2003-03-01), pages 75 - 84 *
DATABASE EMBL RICHARD, S. ET AL.: "Isolation and Characterization of a Dehydrin Gene from White Spruce Induced Upon Wounding, Drought and Cold Stresses.", Database accession no. Q9XEL3_PICGL *
DATABASE NCBI [online] Database accession no. AAW59228 *
DATABASE NCBI [online] Database accession no. AAW59233 *
DATABASE NCBI [online] Database accession no. AAW59253 *
DATABASE NCBI 16 March 2007 (2007-03-16), Database accession no. CAM58808 *
DATABASE NCBI 24 March 2006 (2006-03-24), GONZALES-MARTINEZ, S.C. ET AL.: "DNA Sequence Variation and Selection of Tag . Single-Nucleotide Polymorphisms at Candidate Genes for Drought-Stress Responses in Pinus taeda.L.", Database accession no. AY867580 *
DATABASE NCBI 27 June 2005 (2005-06-27), Database accession no. DR388209 *
GENETICS, vol. 172, March 2006 (2006-03-01), pages 1915 - 1926 *
PLANT MOLECULAR BIOLOGY, vol. 43, 2000, pages 1 - 10 *

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