WO2003016564A2 - Utilisation d'associations entre polymorphismes du gene sh2 et des caracteristiques de qualite de la graine pour la selection de plantes - Google Patents
Utilisation d'associations entre polymorphismes du gene sh2 et des caracteristiques de qualite de la graine pour la selection de plantes Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- the present invention relates to the field of the selection of plant varieties having improved agronomic characteristics, in particular phenotypic quality characteristics of improved seeds. It relates to the detection of phenotypic characteristics of improved seed quality by analysis of the polymorphism of an Sh2 gene to select plants with improved seed quality as well as to means for the implementation of this detection.
- the grain accumulates sufficient energy reserves to allow germination of the embryo. These reserves are in the form of carbohydrate, protein or oleic reserves. In cereals, the accumulated reserves are mainly of carbohydrate and protein forms. Carbohydrate stores are largely made up of polysaccharides such as starch. Starch is made up of two distinct polysaccharide fractions, amylose and amylopectin. Amylose constitutes the minority fraction of starch, it is a chain of glucose monomers linked in ⁇ 1-4 having less than 1% of branches. Amylopectin represents the majority fraction of starch. It consists of glucose monomers linked in ⁇ 1-4 and is branched to approximately 5% with glucose monomers linked to the main chain by ⁇ 1-6 bonds. Starch represents nearly 85% of the weight of the endosperm of grains, the remaining fraction of energy reserves being essentially made up of proteins. Agronomic and nutritional importance of cereals and starch
- Starch is the major energy storage polysaccharide in plants. It is of particular importance in cereals. Thus, starch from rice, wheat or corn constitutes the main sugar intake in food and feed. The study of grain filling processes, the selection and creation of varieties of cereals with modified starch contents constitute one of the favored axes of research concerning cereals.
- ADP Glucose Pyrophosphorylase (AGPase) is a key enzyme in the biosynthetic pathway of polysaccharides, both in bacteria and in plants. In plants, it makes it possible to dephosphorylate glucose-1-phosphate to ADP-glucose, the constituent monomer of starch.
- the plant AGPase has a complex structure in the form of a heterotetramer composed of two types of similar but distinct subunits.
- the two types of subunits with a molecular weight close to 50 kDa in the endosperm, are encoded, one by the Sh2 gene for Shrunken 2, (Bhave et al., 1990) and the other by the Bt2 gene for Brittle-2, (Bae et al., 1990).
- the genome has polymorphism.
- the main causes of these genetic variabilities are mutations occurring during DNA replication which precedes cell multiplication. It can be the insertion or deletion of one or more nucleotides (grouped under the term indel), or a transition type substitution (substitution of a purine base by another purine base or a pyrimidine base by another pyrimidine base) or transversion (substitution of a purine base by a pyrimidine base or vice versa).
- Polymorphic sites are thus classified into two categories depending on whether it is the substitution of a base
- SNP single nucleotide polymorphism
- indel Insertion-Deletion
- the invention provides for the first time a set of means for selecting plant varieties for their improved phenotypic characteristics of seed quality, by the analysis of polymorphisms newly identified in the Sh2 gene, polymorphisms which are statistically associated with a characteristic. specific phenotypic quality of the seed, or a combination of phenotypic characteristics of the quality of the seed.
- the subject of the invention is the use of a probe or a nucleotide primer in a process for selecting plants having improved phenotypic characteristics of seed quality, characterized in that said probe or said nucleotide primer allows the detection of '' a polymorphic base or of a polymorphic nucleotide sequence defining an allele of a polymorphic site of the Sh2 gene of sequence SEQ ID No 1, said polymorphic base or said polymorphic nucleotide sequence being contained in an acid nucleic acid included in the Sh2 gene, chosen from nucleic acids comprising a polymorphic nucleotide site associated with a characteristic or a combination of phenotypic characteristics linked to the quality of the seed, in particular the number of seeds per ear, the mass of the mature seed , the protein content of the seed, the starch content of the seed, the amylose content of the seed or the protein / starch weight ratio of the seed.
- It also relates to a method for determining the identity of the allele of a polymorphic site within a nucleic acid derived from an Sh2 gene with a view to selecting a plant possessing improved phenotypic characteristics of seed quality. , characterized in that it comprises a step of characterizing the identity of the polymorphic base or of the polymorphic nucleotide sequence present at at least one nucleotide position of said nucleic acid corresponding to at least one of the nucleotides included in a newly polymorphic nucleotide site identified from the Sh2 gene.
- determining the identity of the allele of a polymorphic site or of a combination of polymorphic sites makes it possible to predict the quality phenotype of the seed of the plant analyzed, without requiring direct analysis of the characters. phenotypic themselves.
- the invention also relates to nucleotide probes and primers for determining the allelic form of a polymorphic site of the Sh2 gene, useful in particular as means for determining the identity of the polymorphic base or of the polymorphic nucleotide sequence at the associated polymorphic site to a characteristic or a combination of phenotypic characteristics of seed quality.
- nucleic acid derived from the Sh2 gene and comprising at least one polymorphic site as defined in the present description, as well as recombinant vectors comprising such a nucleic acid.
- Figure 1 represents the nucleotide sequence of the Sh2 gene described by Shaw and Hannah (1992), which is identical to the sequence SEQ ID No. 1 of the sequence listing.
- the first column on the left represents the numbering in nucleotides of the sequence SEQ ID No. 1 of the sequence listing of this description.
- the first nucleotide is numbered 1.
- the second column on the left represents the nucleotide numbering prescribed by Shaw and Hannah (1992), which is based on the position of the nucleotide of the transcription initiation site, numbered +1.
- the nucleotide at position 1 of the sequence SEQ ID No. 1 is the nucleotide at position -1020 according to the nomenclature of Shaw and Hannah (1992).
- the Applicant has identified, in the sequence of the Sh2 gene, a collection of polymorphisms which it has shown that they are each associated with at least one phenotypic characteristic defining the quality of the seed of a plant.
- the identification of these polymorphisms has made it possible to develop methods for detecting these polymorphisms from the DNA of a plant individual, plant cell, plant at the seedling stage, plant at an early stage of development or plant at the vegetative stage, making it possible to predict what are or will be the phenotypic characteristics linked to the quality of the seed or of the future seed.
- the association between the presence of a defined allele of a polymorphic site of the Sh2 gene and at least one grain-filling character makes it possible to relate a given allele or a combination of given alleles (haplotype) and a phenotypic character. or a combination of phenotypic traits defining the quality of the seed.
- haplotype a combination of given alleles
- phenotypic traits defining the quality of the seed.
- the inventors have shown that the polymorphism of the Sh2 gene is statistically associated with quality characteristics of the seed, such as:
- the identification of particular alleles of polymorphic sites associated with the quality characteristics of the seed, in the sequence of the Sh2 gene has made it possible to define oligonucleotide sequences specific for a given allele of one or more polymorphic sites of the Sh2 gene. and to use these sequences as probes or primers to facilitate the selection of plants assisted by markers, to generate favorable allelic sequences by site-directed mutagenesis, or to clone sequences bringing a favorable agronomic character, linked to the quality of the seed , to the plant transformed with these sequences. It has also been shown according to the invention that certain polymorphisms of the Sh2 gene cause modifications in the amino acid sequence.
- the favorable alleles identified at these polymorphisms can be used to modify the structure or activity of the AGPase enzyme.
- the Sh2 gene used as the reference nucleotide sequence from which the various newly identified polymorphic sites according to the invention are defined is the nucleotide sequence referenced in the GenBank database under the access number M81603 and which is reproduced as the sequence SEQ ID N ° 1 of the sequence listing.
- the Sh2 gene of sequence SEQ ID No. 1 has 7320 nucleotides in length. It has 16 exons, respectively:
- n ° 8 from the nucleotide in position 3546 to the nucleotide in position 3639 of the sequence SEQ ID N ° 1, which corresponds to the sequence going from the nucleotide in position 2526 to the nucleotide in position 2619 of the Sh2 gene according to the nomenclature of Shaw and Hannah (1992). ;
- the numbering used to define the position of the polymorphic nucleotide or of the polymorphic nucleotide sequence of a polymorphic site of the Sh2 gene is exclusively that described by Shaw and Hannah (1992) and which is recalled in the first column on the left the Sh2 gene sequence shown in Figure 1.
- the polymorphism of the Sh2 gene was determined from DNA from 33 inbred corn lines, whose phenotypic characteristics related to grain quality were simultaneously analyzed.
- polymorphic site of the Sh2 gene is meant a region of the sequence of the Sh2 gene which, in the genome of certain plant lines, more specifically in certain corn lines, differs by one or more consecutive nucleotides with respect to the corresponding region of the Sh2 gene defined by the sequence SEQ ID No. 1.
- a polymorphic site can consist of a variation of a single nucleotide which can take two meanings, for example a base A or a base T, such a polymorphic site being then designated polymorphism SNP (for “Single Nucleotide Polymorphism”).
- a polymorphic site can also consist of the addition or deletion of one or more consecutive nucleotides, relative to the reference sequence SEQ ID No. 1. This second type of polymorphic site is designated "indel” (for "Insertion-Deletion").
- the polymorphic sites of the Sh2 gene characterized in the present invention are defined below. They are characterized by their nucleotide difference compared to the reference Sh2 SEQ ID N ° 1 gene sequence and whose nucleotide positions are defined according to the nomenclature of Shaw and Hannah (1992). As defined below, the polymorphic sites of the Sh2 gene are characterized by their allele, the presence of which, in the genome of a plant, is associated with the expression of a modified phenotypic character of seed quality. . (a) a nucleic acid in which the nucleotide corresponding to the nucleotide in position -921 of the Sh2 gene is a G;
- the presence of a base C constitutes a conservative base substitution causing no change in the amino acid sequence of the corresponding SH2 polypeptide, compared to the SH2 polypeptide of sequence SEQ ID No. 52 coded by the sequence SEQ ID N ° 1.
- the presence of a base C results in the replacement, in the sequence of the SH2 polypeptide of sequence SEQ ID No. 52, of the amino acid Leucine, coded by the sequence SEQ ID No. 1 at this position, by the amino acid Serine.
- the characterization according to the invention of informative polymorphic sites within the sequence of the Sh2 gene has made possible the construction of various means of detection of a given allele of each of the polymorphic sites, useful in particular in selection methods of lines of plants, specifically of corn, having phenotypic characteristics of seed quality sought.
- Such methods of selecting plant lines can be carried out at early stages of plant development, before seed formation, at a time in plant development for which it is not possible to determine the phenotypic characteristics of seed.
- the methods implementing an analysis of the polymorphic sites of the Sh2 gene of the invention therefore have a predictive value of great technical and economic interest.
- the subject of the invention is the use of a probe or a nucleotide primer in a process for selecting plants having improved phenotypic characteristics of seed quality, characterized in that said probe or said nucleotide primer allows the detection of 'a polymorphic base or of a polymorphic nucleotide sequence defining an allele of a polymorphic site of the Sh2 gene of sequence SEQ ID No 1, said polymorphic base or said polymorphic nucleotide sequence being contained in a nucleic acid included in a Sh2 gene, chosen from the following nucleic acids:
- nucleic acid comprising the insertion of the sequence 5'-GTTTTTATTTA-3 'after the nucleotide corresponding to the nucleotide in position +3123 of the Sh2 gene.
- the expression “grain quality” includes the characters influencing the quality and the quantity of the seed such as for example: the number of seeds per ear, the mass of mature seeds, the content of protein, starch, amylose and the weight ratio protein content / starch content in the seed, as well as all the complex characters including one or more of these characters.
- One of the methods which can be used to determine the phenotype of an individual at this or these sites may consist in: i) obtaining a DNA sample from an individual, ii) identify the allele at one or more of the positions mentioned above (with reference to the positions described according to the Genbank sequence no. M81603) in the Sh2 gene, iii) predict the quality of the grain of the individual with reference to l association of the Sh2 gene polymorphism as described above with the grain quality trait.
- the DNA sample is obtained according to conventional methods of DNA extraction used in plants, in particular from young corn leaves (Dellaporta et al. 1983).
- the DNA sample must contain at least the gene sequence
- Sh2 that is to say a region of this sequence which can be amplified by any technique known from the prior art, such as for example, PCR.
- the quality of the grain of the individual studied can be determined by reference to the presence of an allele at at least one, several or all of the positions described in combination with other polymorphisms in the Sh2 gene which are known or which will be characterized. later
- mini-sequencing APEX (Arrayed primer extension), RFLP (Restriction fragment length polymorphism), PCR product restriction sites (CAPS), OLA (Oligonucleotide ligation assay), pyrosequencing (Nyrèn et al. 1997).
- FRET Fluorescence resonance energy transfer
- fluorescence quenching fluorescence polarization
- fluorescence polarization UK 2228998, Zeneca Itd
- chemiluminescence electrochemiluminescence
- radioactivity colorimetry
- hybridization protection assay mass spectrometry.
- Other amplification techniques such as SSR (Self sustained replication), LCR (Ligase chain reaction), SDA (Strand displacement amplification) or leb-DNA (branched DNA).
- SSR Self sustained replication
- LCR Low-Ligase chain reaction
- SDA String displacement amplification
- leb-DNA branched DNA
- said probe or said nucleotide primer can be characterized in that it makes it possible to discriminate between the presence of a first acid nucleic acid (1) and a second nucleic acid (2), said nucleic acids (1) and (2) being chosen from the following: Polymorphic sites
- Site - 921 the nucleic acid (1) of sequence SEQ ID No 2 in which the nucleotide at position 41 is a base G and the nucleic acid (2) of sequence SEQ ID No 2 in which the nucleotide at position 41 is a base A;
- Site - 438 the nucleic acid (1) of sequence SEQ ID No. 3 in which the nucleotide in position 41 is a base G and the nucleic acid (2) of sequence SEQ ID No. 3 in which the nucleotide at position 41 is a base A;
- Site - 362 the nucleic acid (1) of sequence SEQ ID No. 4 in which the nucleotide at position 41 is a base A and the nucleic acid (2) of sequence SEQ ID No. 4 in which the nucleotide at position 41 is a base G;
- Site - 347 the nucleic acid (1) of sequence SEQ ID No. 5 in which the nucleotide at position 41 is a base T and the nucleic acid
- Site - 296 the nucleic acid (1) of sequence SEQ ID No. 6 in which the nucleotide in position 41 is a base T and the nucleic acid (2) of sequence SEQ ID No. 6 in which the nucleotide at position 41 is a base C;
- Site - 277 the nucleic acid (1) of sequence SEQ ID No. 7 in which the nucleotide at position 41 is a base T and the nucleic acid (2) of sequence SEQ ID No. 7 in which the nucleotide at position 41 is a base C;
- Site-266 the nucleic acid (1) of sequence SEQ ID No 8 in which the nucleotide at position 41 is a base C and the nucleic acid (2) of sequence SEQ ID No 8 in which the nucleotide at position 41 is a base T
- Site - 168 the nucleic acid (1) of sequence SEQ ID No 9 in which the nucleotide in position 41 is a base A and the nucleic acid (2) of sequence SEQ ID No 9 in which the nucleotide at position 41 is a base G
- Site - 15 the nucleic acid (1) of sequence SEQ ID No. 10 in which the nucleotide at position 41 is a base A and the nucleic acid (2) of sequence SEQ ID No. 10 in which the nucleotide at position 41 is a base G;
- Site + 35 the nucleic acid (1) of sequence SEQ ID No 11 in which the nucleotide at position 41 is a base T and the nucleic acid (2) of sequence SEQ ID No 11 in which the nucleotide at position 41 is a base C;
- (k) Site + 515 the nucleic acid (1) of sequence SEQ ID No 12 in which the nucleotide at position 41 is a base C and the nucleic acid (2) of sequence SEQ ID No 12 in which the nucleotide at position 41 is a base T;
- Site + 587 the nucleic acid (1) of sequence SEQ ID No. 13 in which the nucleotide at position 41 is a base C and the nucleic acid
- Site + 1059 the nucleic acid (1) of sequence SEQ ID No 16 in which the nucleotide in position 41 is a base G and the nucleic acid (2) of sequence SEQ ID No 16 in which the nucleotide at position 41 is a base C;
- Site + 1068 the nucleic acid (1) of sequence SEQ ID No. 17 in which the nucleotide in position 41 is a base G and the nucleic acid (2) of sequence SEQ ID No. 17 in which the nucleotide at position 41 is a base T;
- Site + 1473 the nucleic acid (1) of sequence SEQ ID No 18 in which the nucleotide at position 41 is a base C and the nucleic acid (2) of sequence SEQ ID No 18 in which the nucleotide at position 41 is a base T;
- Site + 1867 the nucleic acid (1) of sequence SEQ ID No. 19 in which the nucleotide at position 41 is a base C and the nucleic acid
- (t) Site + 2983 the nucleic acid (1) of sequence SEQ ID No. 21 in which the nucleotide at position 41 is a base C and the nucleic acid
- Site + 304 the nucleic acid (1) of sequence SEQ ID No. 27 and the nucleic acid (2) of sequence SEQ ID No. 26;
- Site + 1081 the nucleic acid (1) of sequence SEQ ID No. 29 and the nucleic acid (2) of sequence SEQ ID No. 28;
- Site + 1542 the nucleic acid (1) of sequence SEQ ID No. 33 and the nucleic acid (2) of sequence SEQ ID No. 32;
- Site + 2514 the nucleic acid (1) of sequence SEQ ID No 35 and the nucleic acid (2) of sequence SEQ ID No 34;
- said nucleic acid is a nucleic acid which hybridizes specifically with a nucleic acid of sequence complementary to any one of the nucleic acids (1) or (2) defined in (a) to (ac) above.
- the above use can also be characterized in that: a) the nucleotide probe hybridizes specifically with a nucleic acid of a first allelic form of the polymorphic base or of the polymorphic nucleotide sequence defining a first allele of a polymorphic site of the Sh2 gene and does not hybridize with an acid nucleic acid of a second allelic form of the polymorphic base or of the polymorphic nucleotide sequence defining a second allele of a polymorphic site of the Sh2 gene; or b) the nucleotide primer specifically hybridized with a nucleotide sequence contained in a Sh2 gene, said nucleotide sequence being located upstream of an allelic form of a polymorphic base or of a polymorphic nucleot
- the polymorphism of the Sh2 gene once identified by one of the methods described above can be used, according to the invention, to carry out a predictive selection of plants with better grain quality, including the selection of plants at the seedling stage. and / or at the early stage and / or at the vegetative stage.
- the nucleotide sequence of the corn Sh2 gene SEQ ID No. 1 has a strong nucleotide identity with the nucleotide sequences of many cereals, in particular many varieties of grasses, an identity which is almost complete in the open reading frame (ORF).
- the sequence of the corn Sh2 gene has a very large nucleotide identity with the sorghum Sh2 gene, the greater identity between the two sequences being found in the open reading frame (ORF).
- the SH2 polypeptide encoded by the sorghum Sh2 gene has a difference of a single amino acid with the SH2 polypeptide encoded by the corn Sh2 gene SEQ ID No. 1.
- the inventors believe that the polymorphic sites found in the Sh2 gene of corn are also found in the Sh2 gene of the genome of other cereals, in particular grasses, and even more specifically in the Sh2 gene of sorghum.
- the polymorphic sites located in the open reading frame of the corn Sh2 gene SEQ ID No. 1 are those which have statistically most likely to also be found in the Sh2 genes of other cereals, especially other grasses, such as sorghum.
- Predictive selection therefore applies in particular to cereals such as, for example, corn and sorghum.
- allelic polymorphism described in the invention applies particularly to the selection of varieties of corn.
- SAM marker-assisted selection
- Another advantage of SAM compared to phenotypic evaluation is that the analyzes are completely independent of climatic hazards.
- the above use is further characterized in that the improved phenotypic quality characteristics of the seed are selected from the number of seeds per ear, the mass of the seeds, the protein content of the seeds, the starch content of the seeds. , the amylose content of the seeds and the protein / starch weight ratio of the seeds, or a combination of these phenotypic characteristics.
- a subject of the invention is also a method for determining the identity of the allele of a polymorphic site within a nucleic acid derived from an Sh2 gene with a view to selecting a plant having improved phenotypic quality characteristics.
- the seed characterized in that it comprises a step of characterizing the identity of the polymorphic base or of the polymorphic nucleotide sequence present at at least one nucleotide position of said nucleic acid corresponding to at least one of the nucleotides in position - 921, -830 to -824, -580 to -573, -438, - 362, -347, -296, -277, -266, -168, - 15, +35, +304, +515, +587, +678 , +960, +1059, +1068, +1081, +1473, +1505, +1542, +1867, +2514, +2771, +2939, +2983 and +3123 of the Sh2 gene of sequence SEQ ID No.
- the characterization of the identity of the polymorphic site is carried out by hybridization of a nucleotide probe hybridizing specifically with the sequence of a determined allele of the Sh2 gene.
- the characterization of the identity of the polymorphic site is carried out by hybridization of a nucleotide probe hybridizing specifically with a polymorphic base or a polymorphic nucleotide sequence defining an allele of a determined polymorphic site of the Sh2 gene.
- the characterization of the polymorphic site is carried out by elongation of a nucleotide primer which hybridizes specifically with a nucleotide sequence located upstream of an allelic form of a polymorphic base or of a sequence polymorphic nucleotide defining an allele of a determined polymorphic site of a Sh2 gene.
- the characterization of the identity of the polymorphic site is carried out by hybridization of a nucleotide primer which hybridizes specifically with the sequence situated upstream, on the 5 ′ side of the polymorphic base or of the sequence polymorphic nucleotide defining a given allele of a polymorphic site of the Sh2 gene, then elongation of the primer.
- the characterization of the polymorphic allele can be carried out by sequencing the product of the extension of the primer.
- the nucleotide located at the 3 'end of the primer hybrid with the nucleotide located immediately upstream from the 5' side of the polymorphic base or of the polymorphic nucleotide sequence.
- the identity of the allele of the polymorphic site considered can be determined directly by carrying out the step of elongation of the primer in the presence of fluorescent dideoxynucleotides blocking the elongation reaction.
- the identity of the dideoxynucleotide added to the primer sequence, and therefore the identity of the allele of the polymorphic site, is determined directly by fluorescence analysis. This is the micro-sequencing technique, well known in the state of the art.
- the primer hybrid with the DNA strand comprising the sequence coding for the SH2 polypeptide (the + strand) or with the complementary DNA strand, which carries a base complementary to the corresponding base carried by the “+” strand at the polymorphic site.
- the method is characterized in that in order to select a plant having a modified number of seeds, the identity of the base or of a sequence of bases present at at least one nucleotide position of said nucleic acid corresponding to is determined. at least one of the nucleotides in position -168, +1473, +1542 and +2983 of the Sh2 gene of sequence SEQ ID No. 1.
- the method is characterized in that in order to select a plant with a modified mass of seed, the identity of the base or of a sequence of bases present at at least one nucleotide position of said nucleic acid corresponding to is determined. at least one of the nucleotides in position -168, +1473, +1542 and +2983 of the Sh2 gene of sequence SEQ ID No. 1.
- this method is characterized in that in order to select a plant having a modified protein content in the seed, the identity of the base or of a sequence of bases present at at least one nucleotide position of said acid is determined. nucleic acid corresponding to at least one of the nucleotides in position -
- this method is characterized in that in order to select a plant having a modified starch content in the seed, the identity of the base or of a sequence of bases present at at least one nucleotide position of said acid is determined nucleic acid corresponding to at least one of the nucleotides in position -830 to -824, -362, -347, -296, -15, +515, +587, +1068, +1505 and +2939 of the S 72 gene of sequence SEQ ID # 1.
- this method is characterized in that in order to select a plant having a modified amylose content in the seeds, the identity of the base or of a sequence of bases present at at least one nucleotide position of said acid is determined.
- nucleic acid corresponding to at least one of the nucleotides in position - 438, -266, +678, +960, -921, -580 to -573, -277, +35, +304, +1059, +1081, +1867, +2514, +2771 and +3123 of the Sh2 gene sequence SEQ ID N ° 1.
- this method is characterized in that to select a plant having a modified protein / starch ratio in the seed, the identity of the base or of a sequence of bases present at at least one nucleotide position of said said is determined. nucleic acid corresponding to at least one of the nucleotides in position -168, +1473, +1542, +2983, -830 to -824, -362, -347, -296, -15, +515, +587, +1068, +1505 and +2939 of the Sh2 gene of sequence SEQ ID No. 1.
- said method is characterized in that it is carried out on DNA taken from plants at the seedling stage and / or at the early stage and / or at the vegetative stage.
- the plant is a cereal, preferably a straw cereal.
- the plant is chosen from corn and sorghum.
- Nucleotide probes and primers The invention also relates to the primers and probes obtained from the nucleotide sequences described above and specific for an allele of the Sh2 gene.
- One of the applications of the invention consists in using these probes and / or primers to detect the polymorphism of the Sh2 gene at at least one of the polymorphic sites as defined above.
- the probes and primers obtained according to one aspect of the invention can be used as specific markers for an allele of the Sh2 gene.
- the nucleotide primers specific for alleles, or making it possible to discriminate according to known alleles preferably consist of 8 to 40 nucleotides, more precisely from 17 to 25 nucleotides. The production of such primers is known to those skilled in the art.
- primers comprise a sequence entirely complementary to the sequence defining the reference allele or the “variant” allele associated with a phenotypic character of improved quality of the seed, as defined previously in the description.
- the primers objects of the invention thus produced bear one or more markings to facilitate their detection.
- the labeling can be fluorimetric (digoxygenin, fluorescein, etc.), radioactive (for example 32 P), enzymatic (peroxidase, alkaline phosphatase, etc.), or produced by microbeads of gold, colored glass or plastic.
- nucleic acids according to the invention are useful for the manufacture of probes or primers allowing, when they are used in hybridization, elongation or amplification reactions, to discriminate between them the two alleles of a given polymorphic site of the Sh2 gene characterized according to the invention.
- nucleotide probes and primers hybridizing with a nucleic acid chosen from sequences SEQ ID No. 2 to SEQ ID No. 39, or with a nucleic acid of sequence complementary to one of the sequences SEQ ID n ° 2 to SEQ ID N ° 39.
- the subject of the invention is also a probe or a nucleotide primer characterized in that it makes it possible to discriminate between them the different alleles of a polymorphic site at at least one of the positions-921, -830 to
- the invention also relates to the use of a probe or a primer as defined above as a marker for at least one polymorphic site of the Sh2 gene.
- the invention also relates to diagnostic kits comprising nucleotide sequences as defined above, specific for the favorable allele of the Sh2 gene for one or more of the agronomic characters chosen from the number of grains per ear, the mass of the mature grain , the starch, amylose or protein contents of the grain. It also relates to a kit or kit predicting the phenotypic quality characteristics of a plant seed, characterized in that it comprises: a) a probe or a plurality of probes or primers as defined above; b) where appropriate, the reagents necessary for carrying out a hybridization or amplification reaction.
- the detection kit or kit is characterized in that the probe or probes are immobilized on a support.
- the detection kit or kit is characterized in that the oligonucleotide probes comprise a detectable marker.
- a primer or a nucleotide probe according to the invention can be prepared by any suitable method well known to those skilled in the art, including by cloning and action of restriction enzymes or also by direct chemical synthesis according to techniques such as the method to the phosphodiester of Narang et al. (1979) or Brown et al.
- nucleic acids according to the invention can be labeled, if desired, by incorporating a detectable molecule, that is to say a marker detectable by spectroscopic, photochemical means. , biochemical, immunochemical or even chemical.
- markers can consist of radioactive isotopes ( 32 P 3 H, 35 S), fluorescent molecules (5-bromodeoxyuridine, fluorescein, acetylaminofluorene) or also ligands such as biotin.
- the labeling of the probes is preferably done by incorporating labeled molecules within the polynucleotides by extension of primers, or else by adding to the 5 ′ or 3 ′ ends.
- the probes according to the invention can have structural characteristics such as to allow amplification of the signal, such as the probes described by Urdea et al;
- oligonucleotide probes according to the invention can be used in particular in Southern type hybridizations to the DNA of the Sh2 gene or else in hybridizations to the messenger RNA of this gene when the expression of the corresponding transcript is sought in a sample.
- the probes according to the invention can also be used for the detection of PCR amplification products or even for the detection of mismatches.
- Nucleotide probes or primers according to the invention can be immobilized on a solid support.
- Such solid supports are well known to those skilled in the art and include the surfaces of the wells of micro-titration plates, polystyrene beads, magnetic beads, nitrocellulose strips or even microparticles such as latex particles.
- Means for expression of nucleic acids comprising a polymorphic site of the Sh2 gene.
- the invention also relates to the use of the methods described above for isolating and / or cloning the particular and / or favorable allelic forms of the Sh2 gene, in particular in combination with molecular biology techniques such as for example PCR, RT- PCR, allele sequencing (for example pyrosequencing, ).
- molecular biology techniques such as for example PCR, RT- PCR, allele sequencing (for example pyrosequencing, ).
- a nucleic acid comprising the complete sequence of a determined allele of the Sh2 gene and comprising at least one polymorphic base of a polymorphic site as defined in the present description, or a nucleic acid comprising all of the exons of this determined allele of the Sh2 gene can be obtained according to techniques well known to those skilled in the art, for example by the PCR amplification technique using primers which hybridize specifically with the selected target nucleotide ends of the Sh2 gene, as described in particular in the examples.
- a person skilled in the art can obtain a nucleic acid derived from the Sh2 gene and comprising at least one allele associated with a phenotypic characteristic of quantity or quality of the improved seed of at least one polymorphic site according to the invention by amplifying respectively the 5 ′ and 3 ′ part of the Sh2 gene based on the nucleotide sequence SEQ ID No. 1.
- the subject of the invention is also a recombinant vector into which a nucleic acid has been inserted comprising the complete sequence of a determined allele of the Sh2 gene and comprising at least one polymorphic base of a polymorphic site as defined in the present description, or a nucleic acid comprising all of the exons of this determined allele of the Sh2 gene.
- such a nucleic acid is a nucleic acid derived from the Sh2 gene of sequence SEQ ID No. 1 and comprising at least one polymorphic base or at least one polymorphic nucleotide sequence at at least one of the positions -921, -830 to -824, -580 to - 573, - 438, - 362, - 347, -296, -277, -266, -168, -15, +35, +304, +515, +587, +678, +960 , +1059, +1068, +1081, +1473, +1505, +1542, +1867, +2514, +2771, +2939, +2983 and +3123 of the Sh2 gene of sequence SEQ ID No.1.
- a first nucleic acid which is the subject of the invention is a nucleic acid capable of imparting to a plant, preferably a cereal, in particular to a corn, a modified number of seeds, compared to the reference corn of the Black Mexican Sweet variety described. by Shaw and Hannah (1992), said nucleic acid comprising the allelic form associated with the expression of the modified phenotypic character of seed quality, as defined in the present description, at least one polymorphic site chosen from the polymorphic sites - 168 , + 1473, + 1542 and + 2983 of the Sh2 gene of sequence SEQ ID No. 1.
- a second nucleic acid which is the subject of the invention is a nucleic acid capable of imparting to a plant, preferably a cereal, in particular to a corn, a modified mass of the seed compared to the reference corn of the Black Mexican Sweet variety described. by Shaw and Hannah (1992), said nucleic acid comprising the allelic form associated with the expression of the modified phenotypic character of seed quality, as defined in the present description, at at least one polymorphic site chosen from the polymorphic sites - 168. + 1473, + 1542 and + 2983 of the Sh2 gene of sequence SEQ ID No. 1.
- a third nucleic acid which is the subject of the invention is a nucleic acid capable of imparting to a plant, preferably a cereal in particular to a corn, a modified protein content in the seeds, compared to the reference corn of the Black Mexican variety. Sweet described by Shaw and Hannah (1992), said nucleic acid comprising the allelic form associated with the expression of the modified phenotypic character of seed quality, as defined in the present description, at at least one polymorphic site chosen from the sites polymorphs - 168, + 1473, + 1542, + 2983, - 830 to - 824, - 362, - 347, - 296, - 15, + 515, + 1068, + 1505 and + 2939 of the Sh2 gene of sequence SEQ ID N 1.
- a fourth nucleic acid which is the subject of the invention is a nucleic acid capable of imparting to a plant, preferably a cereal in particular to a corn, a modified starch content in the seeds, compared to the reference corn of the Black Mexican variety. Sweet described by Shaw and Hannah (1992), said nucleic acid comprising the allelic form associated with the expression of the modified phenotypic character of seed quality, as defined in the present description, at at least one polymorphic site chosen from the sites polymorphs - 830 to - 824, - 362, - 347, - 296, - 15, + 515, + 587, + 1068, + 1505 and + 2939 of the Sh2 gene of sequence SEQ ID No. 1.
- a fifth nucleic acid which is the subject of the invention is a nucleic acid capable of imparting to a plant, preferably a cereal, in particular to a corn, a modified amylose content in the seeds, compared to the reference corn of the Black variety.
- nucleic acid comprising the allelic form associated with the expression of the modified phenotypic character of seed quality, as defined in the present description, at at least one polymorphic site chosen from the polymorphic sites - 438, - 266, + 678, + 960, - 921, - 580 to - 573, - 277, + 35, + 304, + 1059, + 1081, + 1867, + 2514, + 2771 and + 3123 of Sh2 gene of sequence SEQ ID No. 1.
- a sixth nucleic acid which is the subject of the invention is a nucleic acid capable of conferring on a plant, preferably a cereal in particular on a corn, a protein / starch ratio modified in the seed, compared to the reference corn of the Black Mexican variety.
- nucleic acid comprising the allelic form associated with the expression of the modified phenotypic character of seed quality, as defined in the present description, at at least one polymorphic site chosen from the sites polymorphs-168, + 1473, + 1542, + 2983, - 830 to - 824, - 362, - 347, - 296, - 15, + 515, + 587, + 1068, + 1505 and + 2939 of the sequence Sh2 gene SEQ ID N ° 1.
- the Black Mexican Sweet corn variety described by Shaw and Hannah (1992), which is the reference variety, may also be referred to as "wild" corn variety for the purposes of this description.
- the invention also relates to a recombinant vector, for example a recombinant cloning vector or a recombinant expression vector, into which a nucleic acid as defined above has been inserted.
- a recombinant vector for example a recombinant cloning vector or a recombinant expression vector, into which a nucleic acid as defined above has been inserted.
- the nucleotide sequences constituting particular allelic forms of the Sh2 gene can be cloned, transferred into cells, in particular to produce transgenic plants.
- the nucleotide sequences of the selected loci are introduced into plant cells, either in culture or in plant organs such as, for example, leaves, stems, seeds, roots, etc.
- the natural or synthetic expression of these sequences can be carried out by operably linking these sequences of interest to a promoter, by including the construct in a vector and by introducing the vector into a host cell.
- An endogenous promoter linked to the nucleotide sequence to be introduced can favorably be used.
- the vectors usually used include initiator and terminator sequences for both transcription and translation, and promoters allowing the regulation of the expression of this particular nucleotide sequence.
- the vectors may also include expression cassettes with at least one independent terminator sequence, sequences allowing replication of the cassette in eukaryotes or prokaryotes or both (shuttle vectors) and a selection marker for at least one.
- transcription terminators which can be used, there may be mentioned the polyA 35S terminator of the cauliflower mosaic virus (Franck et al. 1980) or the polyA NOS terminator, which corresponds to the 3 ′ non-coding region of the nopaline synthase gene of the plasmid Ti - Agrobacterium tumefaciens nopaline strain.
- a constitutive promoter allows a strong expression of the transcript in all the tissues of the regenerated plant and will be active in most environmental conditions, and in all of the cellular transformation and differentiation stages.
- an inducible transcription promoter sequence capable of being controlled by environmental conditions or the development stage, such as, for example, the phenylalanine ammonia lyase (PAL), HMG-CoA reductase (HMG) promoters. ), chitinases, glucanases, proteinase inhibitors (PI), genes of the PR1 family, nopaline synthase (nos) or the vspB gene (US-5,670,349), all of these promoters being recalled with the references of the corresponding publications by Table 3 of US Pat. No. 5,670,349.
- PAL phenylalanine ammonia lyase
- HMG HMG-CoA reductase
- PI proteinase inhibitors
- tissue specific promoters such as for example seed specific tissue promoters (Datla, R. et al., 1997), in particular napine promoters (EP-0.255.378), glutenin, heliantinin (WO-92/17580), albumin (WO- 98/45460), oleosin (WO-98/45461), I ⁇ TS1 or I ⁇ TS3 (WO- 99/20775 ).
- the most common methods for introducing nucleic acids into bacterial cells can be used in the context of this invention. It can be the fusion of receptor cells with bacterial protoplasts containing DNA, electroporation, projectile bombardment, infection with viral vectors, etc.
- Bacterial cells are often used to amplify the number of plasmids containing the construct comprising the nucleotide sequence, object of the invention.
- the bacteria are cultured and the plasmids are then isolated according to methods well known to those skilled in the art (see the manuals of protocols already cited), including the plasmid purification kits sold commercially such as EasyPrepI from Pharmacia Biotech or QIAexpress Expression System from Qiagen.
- the plasmids thus isolated and purified are then manipulated to produce other plasmids which will be used to transfect plant cells.
- the transformation of plant cells can be carried out by various methods such as, for example, the transfer of vectors mentioned above in plant protoplasts after incubation of the latter in a polyethylene glycol solution in the presence of divalent cations (Ca 2+), electroporation (Fromm et al. 1985), the use of a particle gun, or micro -cytoplasmic or nuclear injection (Neuhaus et al, 1987).
- various methods such as, for example, the transfer of vectors mentioned above in plant protoplasts after incubation of the latter in a polyethylene glycol solution in the presence of divalent cations (Ca 2+), electroporation (Fromm et al. 1985), the use of a particle gun, or micro -cytoplasmic or nuclear injection (Neuhaus et al, 1987).
- One of the methods of transformation of plant cells which can be used in the context of the invention is the infection of plant cells by a bacterial cell host comprising the vector containing the sequence of interest.
- the cell host can be Agrobacterium tumefaciens (An et al. 1986), or A. rhizogenes (Guerche et al. 1987).
- the transformation of plant cells is carried out by the transfer of the T region of the extrachromosomal circular plasmid inducing Ti tumors of A tumefaciens, using a binary system (Watson et al., 1994).
- a binary system Wang et al., 1994.
- two vectors are constructed. In one of these vectors, the T-DNA region was deleted, with the exception of the right and left edges, a marker gene being inserted between them to allow selection in plant cells.
- the other partner in the binary system is a helper Ti plasmid, a modified plasmid which no longer has T-DNA but still contains the vir virulence genes necessary for the transformation of the plant cell. This plasmid is maintained in Agrobacterium.
- the method described by Ishida et al. (1996) can be applied for the transformation of monocots, in particular maize.
- the transformation is carried out according to the method described by Finer et al. (1992) using the tungsten or gold particle gun.
- the subject of the invention is also the use of a nucleic acid derived from the Sh2 gene of sequence SEQ ID No. 1 as defined above to transform a host cell.
- the host cell is a bacterial host cell, for example an Agrobacterium tumefaciens cell, or a plant cell, preferably a cereal cell, and most preferably a corn, rice, wheat cell, rye or barley.
- the invention also relates to the transformed cells, with the nucleotide sequences constituting particular allelic forms of the Sh2 gene, including microorganisms (viruses and bacteria) and plant cells, in particular corn cells. It relates in particular to a host cell transformed by a nucleic acid derived from the Sh2 gene or by a recombinant vector, as defined above.
- the invention also relates to plants regenerated from the transformed plant cells described above, as well as plants of which at least one of the parents has been regenerated from a transformed plant cell comprising at least one of the nucleotide sequences constituting a form. particular allelic of the Sh2 gene.
- the invention also relates to the use of a nucleic acid, or of a recombinant vector or of a transformed host cell, defined according to the invention, for manufacturing a transformed plant capable of producing seeds with improved industrial or agrifood qualities. .
- a transformed plant comprising a plurality of transformed host cells according to the invention.
- the invention also relates to a process for obtaining a transformed plant capable of producing seeds with improved industrial or agrifood qualities, characterized in that it comprises the following steps: a) transformation of at least one cell vegetable by a nucleic acid or by a recombinant vector according to the invention; b) selection of the transformed cells obtained in step a) having integrated into their genome at least one copy of a nucleic acid according to the invention; c) regeneration of a transformed plant from the transformed cells obtained in step b).
- the invention extends to a transformed plant or any part of a transformed plant as defined in the present description, such as the root, but also the aerial parts such as the stem, the leaf, the flower and especially the seed.
- the invention also relates to a seed or a plant seed produced by a transformed plant as defined above.
- a transformed seed or such a transformed grain comprises one or more cells comprising in their genome one or more copies of a nucleic acid defined according to the invention, if necessary in a controlled and inducible manner.
- Also part of the invention is any transformation product of a seed as defined in the present description.
- the invention also relates to the monoclonal or polyclonal antibodies specifically recognizing polypeptides corresponding to alleles of the Sh2 gene, these alleles comprising at least one of the forms described in positions Nos. -921, -830 to -824, -580 to -573, -438, -362, -347, -296, -277, -266, -168, -15, 35, 304, 515, 587, 678, 960, 1059, 1068, 1081, 1473, 1505, 1542, 1867, 2514, 2771, 2939, 2983, 3123.
- Preferred antibodies according to the invention are the following antibodies:
- the antibodies specifically recognizing the amino acid region of an SH2 polypeptide encoded by the nucleotides located near the +678 polymorphic site, these antibodies being capable of discriminating between the presence of an Alanine residue and the presence of a residue Threonine encoded by the codon comprising the polymorphic base of this polymorphic site;
- the antibodies specifically recognizing the amino acid region of an SH2 polypeptide encoded by the nucleotides located near the polymorphic site +2983, these antibodies being capable of discriminating between the presence of a Leucine residue and the presence of a residue Serine encoded by the codon comprising the polymorphic base of this polymorphic site
- the invention also relates to the diagnostic kit comprising a mixture of these antibodies.
- the antibodies defined above are useful in particular for predicting the phenotypic characteristics of quantity or quality of the seed, without requiring a long and costly direct, for example biochemical, analysis of these phenotypic characteristics.
- Antibodies against the SH2 polypeptides defined above can be prepared according to standard techniques well known to those skilled in the art.
- antibody within the meaning of the present invention, is meant in particular polyclonal or monoclonal antibodies or fragments (for example the fragments F (ab) ' 2 , F (ab)) or any polypeptide comprising a domain of the antibody initial recognizing the polypeptide or the fragment of target polypeptide according to the invention.
- Monoclonal antibodies can be prepared from hybridomas according to the technique described by Kohler and Milstein (1975)
- the present invention also relates to antibodies directed against a polypeptide as described above or a fragment or a variant thereof, such that produced in the trioma technique or the hybridoma technique described by Kozbor et al. (1983).
- the invention also relates to fragments of single chain Fv antibody (ScFv) as described in US Patent No. 4,946,778 or by Martineau et al. (1998).
- the antibodies according to the invention also include fragments of antibodies obtained using phage libraries as described by Ridder et al. (1995) or humanized antibodies as described by Reinmann et al. (1997) and Léger et al. (1997).
- the antibody preparations according to the invention are useful in immunological detection tests intended for the identification of the presence and / or the amount of a SH2 polypeptide as defined above or of a peptide fragment of that - present in a sample.
- An antibody according to the invention may also comprise an isotopic or non-isotopic detectable marker, for example fluorescent, or else be coupled to a molecule such as biotin, according to techniques well known to those skilled in the art.
- the subject of the invention is also a method for detecting the presence of a polypeptide in accordance with the invention in a sample, said method comprising the steps of: a) bringing the sample to be tested into contact with an antibody such as described above; b) detecting the antigen / antibody complex formed.
- the invention also relates to a kit or diagnostic kit for detecting the presence of a polypeptide according to the invention in a sample, said kit comprising: a) an antibody as defined above; b) where appropriate, one or more reagents necessary for the detection of the antigen / antibody complex formed.
- the invention also relates to a database comprising at least any one of the nucleotide sequences described, obtained, isolated or identified in at least one of the applications of the invention.
- DNA was isolated from young leaves by a method described by Causse et al. (1995). Two overlapping fragments were amplified by PCR using primers drawn from the reference sequence of the Sh2 gene in the Black Mexican Sweet SEQ ID No. 1 line (Shaw and Hannah, 1992).
- the first fragment (Sh2-I) of length close to 2653 bp covers approximately 1000bp upstream of the supposed TATA box, the first three introns, the first three exons as well as part of exon 4.
- the second fragment (SH2 -II) has a length close to 2455 bp, and covers introns 3 to 12, exons 3 to 12 as well as about 30 bases of exon 13.
- the primers used are the following (position on the reference sequence) : for Sh2-I:
- the PCR amplifications were carried out in 100 ⁇ l (approximately 0.1 to 1.5 ⁇ g of DNA, 1.75u of Roche taq polymerase Expand High Fidelity, 0.5 ⁇ M of each primer, 0.3 ⁇ M dNTP, 1.875 mM Mg 2+ ) under the following conditions:
- the PCR products were sequenced by the companies Génome Express or Genaxis. First, the PCR primers were used for the sequence, then the central part of the fragments was sequenced using the following new primers: - For Sh2-1:
- Sequencing Analysis and Sequence Navigator from ABI The sequences were aligned either visually or using the alignment procedure Multiple Clustal of Sequence Navigator software. The fragments were sequenced on only one strand. Nevertheless, verifications have been carried out. First of all, the overlapping part of the sequence fragments never showed any inconsistency when performing the contigs. In addition, PCR amplification and sequencing were repeated over a total of around 10000bp in order to verify all of the singleton polymorphisms (case where only one line differs from all the others). Identical results have always been obtained for the two repetitions. The sites for which differences have been observed among the sequences of Sh2 are identified by their position relative to the reference sequence of the lines Black Mexican Sweet SEQ ID No.
- Table 2 allelic forms with the 29 polymorphisms described, including 20 SNPs and 9 indels, named by their position on the reference sequence SEQ ID No. 1 of the line Black Mexican Sweet (BMS).
- BMS Black Mexican Sweet
- the haplotypes H1 to H6 correspond to the following lines:
- Example 2 Identification of statistically significant associations between a given allele of polymorphic sites of the Sh2 gene and one or more phenotypic characteristics of the quality of the seed.
- the 33 lines were randomized and repeated in three blocks. Each line was represented, in each block, by one to four plants sown side by side.
- the plants were self-fertilized and the grains harvested at maturity.
- Analyzes of associations with the molecular polymorphisms of Sh2 focused on average values of each character by experimentation. The protein, starch and amylose content of each line were measured manually for the year 1 test (see detailed methods in Sene et al.
- NIRS near infrared reflectance spectrometry
- the polymorphisms described above are statistically associated with grain characteristics such as the number of grains per ear, the mass of the mature grain, the protein, starch and amylose content of the grain, and the ratio of protein content to starch content of the grain. These associations were highlighted by multiple regression tests using SAS software (1990). When a trait is significantly associated with several sites, it is possible to define the proportion of variability of the trait explained by all of the sites involved (R 2 ⁇ , last column of Table 3). For example, we can note that, in the experiment of the first year, the combination of Indel -830 to -824 and SNP -168 allows to explain 64% of the variance of the protein / starch ratio among the set of lines.
- the statistical associations described in the invention allow the use of the SNPs and indels described for the purpose of phenotypic prediction or of improving the genetic value of the grains. corn.
- Table 3 results of association tests by multiple regressions between each characteristic of the grain and the polymorphic sites in the sequence of Sh2 for 25 to 33 lines. The sites indicated are in complete redundancy with other sites along the sequence of Sh2 (see Table 2).
- the columns ni, moyl, n2, and moy2 contain the numbers and the average values of the characteristic for the two groups of lines discriminated by the corresponding polymorphic sites.
- F Fisher test value
- P degree of significance
- R 2 share of phenotypic variation explained by a site
- R 2 ⁇ share of phenotypic variation explained by a group of sites combined by multiple regression.
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Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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EP02772507A EP1421219A2 (fr) | 2001-08-17 | 2002-08-16 | Utilisation d'associations entre polymorphismes du gene sh2 et des caracteristiques de qualite de la graine pour la selection de plantes |
US10/486,968 US20050250205A1 (en) | 2001-08-17 | 2002-08-16 | Use of associations between at least one nucleic sequence polymorphism of the sh2 gene and at least one seed quality characteristic in plant selection methods |
CA002457787A CA2457787A1 (fr) | 2001-08-17 | 2002-08-16 | Utilisation d'associations entre polymorphismes du gene sh2 et des caracteristiques de qualite de la graine pour la selection de plantes |
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FR0110906A FR2828694B1 (fr) | 2001-08-17 | 2001-08-17 | Utilisation d'associations entre au moins un polymorphisme de sequence nucleique du gene sh2 et au moins une caracteristique de qualite de la graine, dans des procedes de selection de plantes |
FR01/10906 | 2001-08-17 |
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1999007841A2 (fr) * | 1997-08-07 | 1999-02-18 | Washington State University Research Foundation | Mutants regulateurs de l'adp-glucose pyrophosphorylase et compositions et methodes afferentes |
WO1999058698A2 (fr) * | 1998-05-14 | 1999-11-18 | University Of Florida | Mutants thermostables d'enzymes de biosynthese d'amidon |
WO2000029597A2 (fr) * | 1998-11-19 | 2000-05-25 | University Of Florida | Genes mutants codant pour l'adp-glucose pyrophosphorylase chez des vegetaux |
-
2001
- 2001-08-17 FR FR0110906A patent/FR2828694B1/fr not_active Expired - Fee Related
-
2002
- 2002-08-16 WO PCT/FR2002/002898 patent/WO2003016564A2/fr not_active Application Discontinuation
- 2002-08-16 EP EP02772507A patent/EP1421219A2/fr not_active Withdrawn
- 2002-08-16 US US10/486,968 patent/US20050250205A1/en not_active Abandoned
- 2002-08-16 CA CA002457787A patent/CA2457787A1/fr not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999007841A2 (fr) * | 1997-08-07 | 1999-02-18 | Washington State University Research Foundation | Mutants regulateurs de l'adp-glucose pyrophosphorylase et compositions et methodes afferentes |
WO1999058698A2 (fr) * | 1998-05-14 | 1999-11-18 | University Of Florida | Mutants thermostables d'enzymes de biosynthese d'amidon |
WO2000029597A2 (fr) * | 1998-11-19 | 2000-05-25 | University Of Florida | Genes mutants codant pour l'adp-glucose pyrophosphorylase chez des vegetaux |
Non-Patent Citations (3)
Title |
---|
GIROUX M J ET AL: "A SINGLE GENE MUTATION THAT INCREASES MAIZE SEED WEIGHT" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 93, no. 12, 1 juin 1996 (1996-06-01), pages 5824-5829, XP000652281 ISSN: 0027-8424 cité dans la demande * |
PRIOUL JEAN-LOUIS ET AL: "From QTLs for enzyme activity to candidate genes in maize." JOURNAL OF EXPERIMENTAL BOTANY, vol. 50, no. 337, août 1999 (1999-08), pages 1281-1288, XP002197282 ISSN: 0022-0957 * |
See also references of EP1421219A2 * |
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WO2003016564A3 (fr) | 2004-02-19 |
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EP1421219A2 (fr) | 2004-05-26 |
US20050250205A1 (en) | 2005-11-10 |
FR2828694A1 (fr) | 2003-02-21 |
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