WO2011162612A1 - Procédé permettant de moduler le niveau de phosphorylation de l'amidon dans une lignée végétale, procédé permettant de sélectionner une plante ou une partie de celle-ci, incluant une graine et un tubercule, et son utilisation - Google Patents

Procédé permettant de moduler le niveau de phosphorylation de l'amidon dans une lignée végétale, procédé permettant de sélectionner une plante ou une partie de celle-ci, incluant une graine et un tubercule, et son utilisation Download PDF

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WO2011162612A1
WO2011162612A1 PCT/NL2011/050460 NL2011050460W WO2011162612A1 WO 2011162612 A1 WO2011162612 A1 WO 2011162612A1 NL 2011050460 W NL2011050460 W NL 2011050460W WO 2011162612 A1 WO2011162612 A1 WO 2011162612A1
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plant
gwd
allele
starch
alleles
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Anna Maria Agnes Wolters
Johannes Gerardus Anna Maria Ludovicus Uitdewilligen
Herman Johannes Van Eck
Nicolaas Clemens Maria Henricus De Vetten
Richard Gerardus Franciscus Visser
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Wageningen Universiteit
Stichting Voor De Technische Wetenschappen
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B31/00—Preparation of derivatives of starch
    • C08B31/08—Ethers
    • C08B31/12—Ethers having alkyl or cycloalkyl radicals substituted by heteroatoms, e.g. hydroxyalkyl or carboxyalkyl starch
    • C08B31/125—Ethers having alkyl or cycloalkyl radicals substituted by heteroatoms, e.g. hydroxyalkyl or carboxyalkyl starch having a substituent containing at least one nitrogen atom, e.g. cationic starch
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L3/00—Compositions of starch, amylose or amylopectin or of their derivatives or degradation products
    • C08L3/04—Starch derivatives, e.g. crosslinked derivatives
    • C08L3/06—Esters
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L3/00—Compositions of starch, amylose or amylopectin or of their derivatives or degradation products
    • C08L3/04—Starch derivatives, e.g. crosslinked derivatives
    • C08L3/08—Ethers
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D103/00—Coating compositions based on starch, amylose or amylopectin or on their derivatives or degradation products
    • C09D103/04—Starch derivatives
    • C09D103/06—Esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1294—Phosphotransferases with paired acceptors (2.7.9)

Definitions

  • step c) selecting progeny from crossing step b) for a genotype wherein allele A of GWD is absent, d) selfing and /or further crossing said progeny selected in step c),
  • the term "nulliplex”, “simplex”, “duplex”, “triplex” and “quadruplex” is defined as a genetic condition existing when a specific allele at a corresponding locus on corresponding homologous chromosomes is present 0, 1 , 2, 3 or 4 times, respectively.
  • the phenotypic effect associated with a recessive allele is only observed when the allele is present in quadruplex condition, whereas the phenotypic effect associated with a dominant allele is already observed when the allele is present in a simplex or higher condition.
  • haplotype means a combination of alleles at multiple loci that are transmitted together on the same chromosome. This includes haplotypes referring to as few as two loci, and haplotypes referring to an entire chromosome depending on the number of recombination events that have occurred between a given set of loci. It also includes a set of polymorphisms on a single chromatid that are statistically associated with each other.
  • primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization.
  • the exact lengths of the primers will depend on many factors, including temperature and source of primer.
  • a "pair of bi-directional primers" as used herein refers to one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification.
  • stringency or “stringent hybridization conditions” refer to hybridization conditions that affect the stability of hybrids, e.g., temperature, salt concentration, pH, formamide concentration and the like. These conditions are empirically optimised to maximize specific binding and minimize non-specific binding of primer or probe to its target nucleic acid sequence.
  • the terms as used include reference to conditions under which a probe or primer will hybridise to its target sequence, to a detectably greater degree than other sequences (e.g. at least 2-fold over background).
  • Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridise specifically at higher temperatures.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridises to a perfectly matched probe or primer.
  • the present invention now enables a method for increasing the degree of phosphorylation of starch in a plant comprising:
  • the invention provides a method for increasing the degree of phosphorylation in starch in a plant, comprising enriching a plant for the presence of the alleles C or H of GWD by
  • step b) selecting progeny from crossing step b) for a genotype wherein at least one of alleles C or H of GWD is present,
  • step d) selecting progeny resulting from step d) for a genotype wherein at least one of alleles C or H of GWD is present, and
  • StPho2 starch phosphorylase 2
  • allele 2 of StPho2 was correlated with a high amount of starch phosphate
  • allele 1 was correlated with a low amount of starch phosphate.
  • StPho2 allele 2 can be distinguished from other StPho2 alleles by sequencing the PCR product obtained with primers Pho2-lF (5 ' -GAAGATGGAAAGGGTTCTC A-3 ') and Pho2-lR (5'- TTAGCCATATGCACAACAGG-3 ' ) .
  • step c) selfing and/or further crossing said progeny selected in step c),
  • phosphorylase 2 (StPho2) may be obtained.
  • a simultaneous enrichment for allele 2 of starch phosphorylase 2 (StPho2) and for any of alleles C or H of the GWD gene may be used for obtaining an additional enrichment effect.
  • a positive selection for allele 2 of starch phosphorylase 2 (StPho2) may be combined with a negative selection for allele A of GWD.
  • the application provides for a method of producing the plants, by involving them in a breeding programme, either the programme for enriching for the C and/or H alleles, and/or the programme for decreasing the amount of A alleles, as mentioned above, select the plants of interest and grow these plants.
  • Selected plants that are used for crossing purposes in the methods according to the invention may have any type of ploidy.
  • selected plants may be haploid, diploid or tetraploid.
  • crossing diploid plants will only provide diploid offspring.
  • Crossing a diploid plant with a tetraploid plant will result in triploid offspring that is sterile.
  • selected plants are crossed with each other using classical in vivo crossing methods that comprise one or more crossing steps including selfing.
  • classical crossing steps characteristics of both the parents can be combined in the progeny.
  • a plant that provides a high yield can be crossed with a plant that contains large amounts of a certain nutrient.
  • Such a crossing would provide progeny comprising both characteristics, i.e. plants that not only comprise large amounts of the nutrient but also provide high yields.
  • the inventors now have identified two regions in the genomic sequence of the glucan water dikinase (GWD) gene containing SNPs that enable discrimination between 10 different alleles for the gene. These regions are a large part (627 basepairs) of the region exon 8 to exon 9 of the gene (hereinafter identified as GWDex7) and a region (606 basepairs) covering exon 15 to exon 17 (hereinafter identified as GWD56).
  • GWDex7 region exon 8 to exon 9 of the gene
  • GWD56 region (606 basepairs) covering exon 15 to exon 17
  • Allele B as compared to allele Al differs in many places in the GWDex7 area: positions 139 (A ⁇ T), 237 (T ⁇ C), 261 (C ⁇ T), 268 (G ⁇ C), 271 (T ⁇ C), 283 (T ⁇ G), 292 (T ⁇ G), 448 (C ⁇ T), 459
  • allele B the GWDex7 area is unique with respect to all other alleles by the T460A mutation. Further, as can be seen, allele B differs from allele Al in 8 positions in the GWD56 area. In the present application, the alleles will be indicated by their letter as used in the below Table.
  • Table 1 Single nucleotide polymorphisms in the areas GWDex7 and GWD56:
  • Methods for detecting a nucleotide change can utilize one or more oligonucleotide probes or primers that selectively hybridize to a target polynucleotide which contains one or more SNP positions or other markers.
  • probes or primers include, for example, an amplification primer pair.
  • Probes useful in practicing a method of the invention can include, for example, an oligonucleotide that is complementary to and spans a portion of the target polynucleotide, including the position of the marker, wherein the presence or absence of a specific nucleotide at the position (e.g, an SNP, an indel or a transposon insertion) is detected by the presence or absence of selective hybridization of the probe.
  • a specific nucleotide at the position e.g, an SNP, an indel or a transposon insertion
  • Such a method can further include contacting the target polynucleotide and hybridized oligonucleotide with an endonuclease, and detecting the presence or absence of a cleavage product of the probe, depending on whether the nucleotide occurrence at the marker site is complementary to the corresponding nucleotide of the probe.
  • a pair of probes that specifically hybridize upstream and adjacent and downstream and adjacent to the site of the marker, wherein one of the probes includes a nucleotide complementary to a nucleotide occurrence of the marker also can be used in an oligonucleotide ligation assay, wherein the presence or absence of a ligation product is indicative of a specific nucleotide occurrence at the marker site.
  • An oligonucleotide also can be useful as a primer, for example, for a primer extension reaction, wherein the product (or absence of a product) of the extension reaction is indicative of the nucleotide occurrence.
  • polynucleotide including the marker site can be useful, wherein the amplification product is examined to determine the nucleotide occurrence at the marker site.
  • nucleotide occurrence of a marker is such that the nucleotide occurrence results in an amino acid change in an encoded polypeptide
  • nucleotide occurrence can be identified indirectly by detecting the particular amino acid in the polypeptide.
  • the method for determining the amino acid will depend, for example, on the structure of the polypeptide or on the position of the amino acid in the polypeptide.
  • the polypeptide contains only a single occurrence of an amino acid encoded by the particular polymorphism, the polypeptide can be examined for the presence or absence of the amino acid.
  • the amino acid is at or near the amino terminus or the carboxy terminus of the polypeptide
  • simple sequencing of the terminal amino acids can be performed.
  • the polypeptide can be treated with one or more enzymes and a peptide fragment containing the amino acid position of interest can be examined, for example, by sequencing the peptide, or by detecting a particular migration of the peptide following electrophoresis.
  • the particular amino acid comprises an epitope of the polypeptide
  • the specific binding, or absence thereof, of an antibody specific for the epitope can be detected.
  • Other methods for detecting a particular amino acid in a polypeptide or peptide fragment thereof are well known and can be selected based, for example, on convenience or availability of equipment such as a mass-spectrometer, capillary
  • electrophoresis system magnetic resonance imaging equipment, and the like.
  • the marker-assisted selection steps in the methods of the invention can in principle be performed by applying any nucleic acid amplification method, such as the Polymerase Chain Reaction (PCR; Mullis 1987, U.S. Pat. No. 4,683,195, 4,683,202, en 4,800,159) or by using amplification reactions such as Ligase Chain Reaction (LCR; Barany 1991, Proc. Natl. Acad. Sci. USA 88: 189-193; EP Appl. No., 320,308), Self- Sustained Sequence Replication (3SR; Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), Strand Displacement Amplification (SDA; U.S. Pat. Nos.
  • PCR Polymerase Chain Reaction
  • LCR Ligase Chain Reaction
  • LCR Ligase Chain Reaction
  • SDA Strand Displacement Amplification
  • an amplification reaction may be performed under conditions of reduced stringency (e.g. a PCR amplification using an annealing temperature of 38°C, or the presence of 3.5 mM MgCl 2 ).
  • conditions of reduced stringency e.g. a PCR amplification using an annealing temperature of 38°C, or the presence of 3.5 mM MgCl 2 .
  • the person skilled in the art will be able to select conditions of suitable stringency.
  • the detection of the amplification products can in principle be accomplished by any suitable method known in the art.
  • the detection fragments may be directly stained or labeled with radioactive labels, antibodies, luminescent dyes, fluorescent dyes, or enzyme reagents.
  • Direct DNA stains include for example intercalating dyes such as acridine orange, ethidium bromide, ethidium monoazide or Hoechst dyes.
  • the DNA fragments may be detected by incorporation of labeled dNTP bases into the synthesized DNA fragments.
  • Detection labels which may be associated with nucleotide bases include e.g. fluorescein, cyanine dye or BrdU.
  • a suitable detection procedure for use in the present invention may for example comprise an enzyme immunoassay (EIA) format.
  • EIA enzyme immunoassay
  • Probes useful for the detection of the target DNA as disclosed herein preferably bind only to at least a part of the DNA sequence region as amplified by the DNA amplification procedure. Those of skill in the art can prepare suitable probes for detection based on the nucleotide sequence of the target DNA without undue experimentation. Also the
  • complementary sequences of the target DNA may suitably be used as detection probes in a method of the invention, provided that such a complementary strand is amplified in the amplification reaction employed.
  • Any suitable method for screening the nucleic acids of a plant or part thereof for the presence or absence of polymorphisms is considered to be part of the methods according to the invention.
  • Such screening methods include, but are not limited to: DNA sequencing, restriction fragment length polymorphism (RFLP) analysis, amplified fragment length polymorphism (AFLP) analysis; heteroduplex analysis, single strand conformational polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), real time PCR analysis (e.g.
  • nucleic acid amplification techniques allow the amplification of fragments of nucleic acids, which may be present in very low amounts.
  • the SNP-specific sequences must be determined for which primers or probes may then be developed.
  • the nucleic acid may be isolated from any raw sample material, optionally reverse transcribed into cDNA and directly cloned and/or sequenced.
  • DNA and RNA isolation kits are commercially available from for instance QIAGEN GmbH, Hilden, Germany, or Roche Diagnostics, a division of F. Hoffmann-La Roche Ltd, Basel, Switzerland. Nucleic acid-based detection of insertions or deletions can be accomplished accordingly.
  • a sample useful for practicing a method of the invention can be any biological sample from a plant or a part thereof that contains nucleic acid molecules, including portions of the allele sequences to be examined, or corresponding encoded polypeptides, depending on the particular method.
  • the sample can be a cell or tissue sample.
  • the nucleic acid sample generally is a deoxyribonucleic acid (DNA) sample, particularly genomic DNA or an amplification product thereof.
  • DNA deoxyribonucleic acid
  • RNA hetero-nuclear ribonucleic acid
  • the nucleic acid sample can thus be DNA or RNA, or products derived therefrom such as, for example, amplification products.
  • nucleic acid hybridization probes and/or nucleic acid amplification primers may be designed an used in a detection assay for detecting the above identified SNPs in a sample as defined herein.
  • the DNA, or alternatively, the cDNA may be PCR amplified by using for instance Pfu and Taq DNA polymerases and amplification primers specific for the SNP DNA sequences. Also complete commercially available systems may be used for PCR (e.g. available form various suppliers such as Roche Diagnostics).
  • a suitable method may for instance include mixing into a suitable aqueous buffering system (e.g. a commercially available PCR buffer) a suitable amount of total DNA as a template (e.g. 1 to 5 ⁇ g), a suitable amount (e.g.
  • hybridization signal refers to the amount of amplification product produced upon a certain number of cycles and thus to the amount of target DNA available as template in the reaction.
  • the DNA or RNA fragments may be detected by incorporation of labeled dNTP bases into the synthesized fragments.
  • Detection labels which may be associated with nucleotide bases include e.g. fluorescein, cyanine dye, digoxigenin (DIG) or
  • RNA extension assay a primer extension assay
  • Taqman® PCR a differential hybridization assay
  • an assay which detects allele- specific enzyme cleavage an assay which detects allele-specific enzyme cleavage
  • allele-specific PCR an assay which detects allele-specific enzyme cleavage
  • Probes useful for the detection of the target nucleic acid sequences preferably bind only to at least a part of the nucleic acid sequence region as amplified by the nucleic acid amplification procedure.
  • Those of skill in the art can prepare suitable probes for detection based on the nucleotide sequence of the target nucleic acid without undue experimentation as set out herein.
  • the specific amplicon detection probes may comprise a label moiety such as a fluorophore, a chromophore, an enzyme or a radio-label, so as to facilitate monitoring of binding of the probes to the reaction product of the amplification reaction.
  • labels are well known to those skilled in the art and include, for example, fluorescein isothiocyanate (FITC), ⁇ -galactosidase, horseradish peroxidase, streptavidin,
  • biotin biotin, digoxigenin, S, C, P or I. Other examples will be apparent to those skilled in the art.
  • Detection may also be performed by a so-called reverse line blot (RLB) assay, such as for instance described by Van den Brule et al. (2002).
  • RLB probes are preferably synthesized with a 5' amino group for subsequent immobilization on e.g. carboxyl coated nylon membranes.
  • the advantage of an RLB format is the ease of the system and its speed, thus allowing for high throughput sample processing.
  • nucleic acid probes for the detection of RNA or DNA fragments is well known in the art. Usually these procedures comprise the hybridization of the target nucleic acid with the probe followed by post-hybridization washings. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution.
  • Tm 81.5 °C + 16.6 (log M) + 0.41 (% GQ-0.61 ( form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the nucleic acid, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
  • the Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe.
  • Tm is reduced by about 1 °C for each 1 % of mismatching; thus, the hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with > 90% identity are sought, the Tm can be decreased 10°C. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH.
  • a modulation in the degree of phosphorylation may also achieved by transgenic methods. Again in such a case, for an increase of the degree of phosphorylation in starch an enrichment for the GWD portions C and H and a decrease in the GWD allele A should be sought. Also enrichment for allele 1 of StPho2 is an alternative or additional approach to reach the intended goal.
  • Transgenic approaches can consist of introducing the above-mentioned alleles in a plant, which is a common procedure and which is well known to the skilled person. More appropriate, however, is site directed mutagenesis, in which one or more of the above- mentioned alleles is used to replace the corresponding endogenously present alleles.
  • Methods for site directed mutagenesis of plants are known such as, for instance, using homologous recombination as been described in WO 02/052026 and in a reviw of May, G.D. and Kmiec, E.B., 2000, AgBiotechNet, 2: 1-3.
  • an allele C or H is introduced by exchange with an allele A of GWD by homologous recombination.
  • also introduction of allele 2 of StPho2 by exchange with allele 1 of StPho2 is performed.
  • H7322 H7322, or AM79.7322 originally from G. Wenzel, Institut fur Genetik, Griinbach,
  • AWGWD1 1 GAAGAAATTATACGTGCTGGAT 3033 F exon 22
  • haplotype G was found only in ancient cultivar accessions, introduced between 1804 to 1872, and that haplotype H is relatively new in the analyzed pool (figure 3).
  • haplotype H All analyzed accessions harboring haplotype H have a lineage descending from the USDA 96-56 accession, which has been used for the introgression of the Phytophthora resistance gene Rl from S. demissum (Leonards-Schippers et al. 1992; Ballvora et al. 2002). Also the rare haplotype A (1) is relatively new in the cultivated potato genepool. The first cultivar in our dataset harbouring this haplotype is Maris Piper, introduced in 1963. This is also the first cultivar harbouring the nematode resistance allele HI, providing resistance to Grol (Ellenby 1952; Toxopeus and Huijsman 1953; Ross 1979). The HI resistance was introduced by introgression from S. tuberosum ssp. andigena clone CPC 1673. All accessions in which we could identify haplotype A (1) had this CPC clone as parent in their pedigree and are, according to the European potato database, resistant to Grol .
  • QTL analysis showed three major additive QTLs regions on chromosome 2, 5 and 9 respectively (figure 7).
  • the QTL on chromosome 2 with a LOD score of 6.41, explained 33.8% of the observed variance.
  • the chromosome 5 QTL (LOD 6.06) explained 28.5% of the variance and the chromosome 9 QTL (LOD4.47) explained 23.7% of the variance.
  • StPho2 allele 2 can be distinguished from other StPho2 alleles by sequencing the PCR product obtained with primers Pho2-lF (5 ' -GAAGATGGAAAGGGTTCTCA-3 ' ) and Pho2- 1R (5 ' -TTAGCC ATATGC AC AAC AGG-3 ' ) .
  • the nucleotide at position 278 (counting from the first nucleotide of primer Pho2-lF) is A for allele 2, while it is G for all other alleles (SNP G278A).
  • Starch of the genotypes with the GWD alllele in different dosages is analysed for starch phosphate content. It shows that with decreasing dosage of allele A and with increasing dosage of allele C starch phosphate content increases.
  • the GBSS mutation in potato is similar to the so-called waxy (wx) mutation in maize and prevents the production of amylose, when expression or specific function of the GBSS protein is absent. Therefore, this mutation has been designated as amylose-free (amf) mutant of potato.
  • amf-gene mutation (allele a) stands for a modification of the GBSS-gene that leads to a complete functional loss of GBSS-activity, notwithstanding that GBSS-like gene products, without the specific activity, may still be expressed from the gene's transcripts in question, whereby the Amf-gene (allele A) stands for a gene from which gene products with GBSS-activity can still be obtained.
  • amf-gene character is determined by a monogenic mendelian recessive gene, the phenotype of which can be detected in various plant parts such as columella cells of root tips, tubers, plastids in the stomatal guard cells and in microspores (Jacobsen et al., 1989, Euphytica 44:43-48). When these parts are stained with a potassium iodine solution (Lugol), starch is stained red in mutants and dark blue in the wild type.
  • a potassium iodine solution Ligol
  • tetraploid clones with nulliplex, simplex, duplex, triplex and quadriplex genotypes at the GWD or StPho2 locus were selected in the nulliplex, simplex, duplex, triplex and quadriplex genotypes at the Am -locus.
  • amylose and starch-bound phosphate content in various parts of the mutant potato plant were investigated.
  • triplex/quadruplex for GWD allele C, or H were selectedThis selection was based on High Resolution Melting analysis using the LightScanner (Idaho Technologies, US).
  • Cationic regular potato starch used was Amylofax PW from AVEBE.
  • test pulp consisted of 80 % birch sulphate cellulose fibres (as in Example 1) and of 20 % Hydrocarb 90.
  • the specific conductivity and the water hardness is artificially increased to a level of 3000 ⁇ 8 : ⁇ (sodium sulphate) and 40°GH (calcium chloride)
  • the cationic starch dosage is 0.8 %, with and without 0.3 % as is Paper Pac N (PPN).
  • the zeta-potential is measured and the starch content of the filtrate is determined.
  • the hand sheet tests are performed with the dynamic hand sheet former (FRET).
  • FRET dynamic hand sheet former
  • the machine settings are:
  • the pulp is diluted to 0.25% with water with a similar specific conductivity and water hardness as the test pulp

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Abstract

La présente invention a trait à un procédé permettant de moduler le niveau de phosphorylation de l'amidon chez les plantes en changeant la composition d'allèle du gène alpha-glucane water dikinase (GWD). Une réduction de la quantité (dosage) de l'allèle A et/ou une augmentation de la quantité (dosage) de l'allèle C et/ou H infèrent une augmentation de la teneur en phosphate de l'amidon. Les décalages de la composition d'allèle peuvent être établis au moyen d'une amélioration génétique, telle que l'introgression, ou grâce à un moyen transgénique, tel que la recombinaison homologue.
PCT/NL2011/050460 2010-06-25 2011-06-24 Procédé permettant de moduler le niveau de phosphorylation de l'amidon dans une lignée végétale, procédé permettant de sélectionner une plante ou une partie de celle-ci, incluant une graine et un tubercule, et son utilisation WO2011162612A1 (fr)

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