EP1250440A1 - Identifying genes associated with a qtl corn digestibility locus - Google Patents
Identifying genes associated with a qtl corn digestibility locusInfo
- Publication number
- EP1250440A1 EP1250440A1 EP01903991A EP01903991A EP1250440A1 EP 1250440 A1 EP1250440 A1 EP 1250440A1 EP 01903991 A EP01903991 A EP 01903991A EP 01903991 A EP01903991 A EP 01903991A EP 1250440 A1 EP1250440 A1 EP 1250440A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- digestibility
- corn
- qtl
- coding
- locus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 235000019621 digestibility Nutrition 0.000 title claims abstract description 118
- 235000002017 Zea mays subsp mays Nutrition 0.000 title claims abstract description 74
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 title claims abstract description 53
- 235000005822 corn Nutrition 0.000 title claims abstract description 53
- 240000008042 Zea mays Species 0.000 title claims description 72
- 108090000623 proteins and genes Proteins 0.000 title abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 38
- 230000002759 chromosomal effect Effects 0.000 claims description 42
- 108700028369 Alleles Proteins 0.000 claims description 30
- 241000196324 Embryophyta Species 0.000 claims description 24
- 102000004190 Enzymes Human genes 0.000 claims description 23
- 108090000790 Enzymes Proteins 0.000 claims description 23
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims description 21
- 235000009973 maize Nutrition 0.000 claims description 21
- 210000000349 chromosome Anatomy 0.000 claims description 19
- 101000762164 Arabidopsis thaliana Cytochrome P450 84A1 Proteins 0.000 claims description 18
- 239000003550 marker Substances 0.000 claims description 17
- 101000984031 Aspergillus flavus (strain ATCC 200026 / FGSC A1120 / IAM 13836 / NRRL 3357 / JCM 12722 / SRRC 167) Cytochrome P450 monooxygenase lnaD Proteins 0.000 claims description 16
- 210000004027 cell Anatomy 0.000 claims description 15
- 230000002349 favourable effect Effects 0.000 claims description 15
- 239000000523 sample Substances 0.000 claims description 14
- 230000009466 transformation Effects 0.000 claims description 9
- 102100034581 Dihydroorotase Human genes 0.000 claims description 8
- 102000054766 genetic haplotypes Human genes 0.000 claims description 8
- 108020004707 nucleic acids Proteins 0.000 claims description 7
- 102000039446 nucleic acids Human genes 0.000 claims description 7
- 150000007523 nucleic acids Chemical class 0.000 claims description 7
- 241001057636 Dracaena deremensis Species 0.000 claims description 6
- 101710191326 4-coumarate-CoA ligase 2 Proteins 0.000 claims description 4
- 101150001464 4CL1 gene Proteins 0.000 claims description 4
- 230000008929 regeneration Effects 0.000 claims description 4
- 238000011069 regeneration method Methods 0.000 claims description 4
- 230000009261 transgenic effect Effects 0.000 claims description 4
- 101710191327 4-coumarate-CoA ligase 1 Proteins 0.000 claims description 3
- 101150019620 CAD gene Proteins 0.000 claims description 3
- 108010061190 Cinnamyl-alcohol dehydrogenase Proteins 0.000 claims description 3
- 108090000854 Oxidoreductases Proteins 0.000 claims description 3
- 102000004316 Oxidoreductases Human genes 0.000 claims description 3
- 101150108672 ccr gene Proteins 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 238000003205 genotyping method Methods 0.000 claims description 2
- 238000012544 monitoring process Methods 0.000 claims description 2
- 238000010187 selection method Methods 0.000 claims description 2
- 229920005610 lignin Polymers 0.000 abstract description 15
- 230000015572 biosynthetic process Effects 0.000 abstract description 6
- 238000003786 synthesis reaction Methods 0.000 abstract description 2
- 241000209149 Zea Species 0.000 abstract 2
- 229940088598 enzyme Drugs 0.000 description 18
- 230000002068 genetic effect Effects 0.000 description 12
- 206010020649 Hyperkeratosis Diseases 0.000 description 11
- 239000002609 medium Substances 0.000 description 10
- 239000013598 vector Substances 0.000 description 7
- 230000014509 gene expression Effects 0.000 description 6
- 238000013507 mapping Methods 0.000 description 6
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 6
- 108020004635 Complementary DNA Proteins 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 210000002257 embryonic structure Anatomy 0.000 description 5
- 230000002255 enzymatic effect Effects 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 238000001320 near-infrared absorption spectroscopy Methods 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 4
- GPRBEKHLDVQUJE-VINNURBNSA-N cefotaxime Chemical compound N([C@@H]1C(N2C(=C(COC(C)=O)CS[C@@H]21)C(O)=O)=O)C(=O)/C(=N/OC)C1=CSC(N)=N1 GPRBEKHLDVQUJE-VINNURBNSA-N 0.000 description 4
- 229960004261 cefotaxime Drugs 0.000 description 4
- 210000002421 cell wall Anatomy 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 238000009396 hybridization Methods 0.000 description 4
- 241000282849 Ruminantia Species 0.000 description 3
- 230000008045 co-localization Effects 0.000 description 3
- 239000000470 constituent Substances 0.000 description 3
- 238000005553 drilling Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000005416 organic matter Substances 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 239000004460 silage Substances 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 241001635598 Enicostema Species 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000000408 embryogenic effect Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000004459 forage Substances 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 210000004767 rumen Anatomy 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- NGSWKAQJJWESNS-ZZXKWVIFSA-N trans-4-coumaric acid Chemical compound OC(=O)\C=C\C1=CC=C(O)C=C1 NGSWKAQJJWESNS-ZZXKWVIFSA-N 0.000 description 2
- 108010016192 4-coumarate-CoA ligase Proteins 0.000 description 1
- 101150062300 4CL2 gene Proteins 0.000 description 1
- 241000589158 Agrobacterium Species 0.000 description 1
- 235000011331 Brassica Nutrition 0.000 description 1
- 241000219198 Brassica Species 0.000 description 1
- 101710100330 Caffeic acid 3-O-methyltransferase Proteins 0.000 description 1
- HZZVJAQRINQKSD-UHFFFAOYSA-N Clavulanic acid Natural products OC(=O)C1C(=CCO)OC2CC(=O)N21 HZZVJAQRINQKSD-UHFFFAOYSA-N 0.000 description 1
- 102000005870 Coenzyme A Ligases Human genes 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108010011449 Long-chain-fatty-acid-CoA ligase Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091092878 Microsatellite Proteins 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 241000219000 Populus Species 0.000 description 1
- 241000183024 Populus tremula Species 0.000 description 1
- 240000004923 Populus tremuloides Species 0.000 description 1
- 235000011263 Populus tremuloides Nutrition 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 101100018379 Shigella flexneri icsA gene Proteins 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 101100476911 Yersinia enterocolitica yscW gene Proteins 0.000 description 1
- 208000005652 acute fatty liver of pregnancy Diseases 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 229940098164 augmentin Drugs 0.000 description 1
- 230000010165 autogamy Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- LHJQIRIGXXHNLA-UHFFFAOYSA-N calcium peroxide Chemical compound [Ca+2].[O-][O-] LHJQIRIGXXHNLA-UHFFFAOYSA-N 0.000 description 1
- 108010042238 caspase-activated deoxyribonuclease Proteins 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000008094 contradictory effect Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000008121 plant development Effects 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000010845 search algorithm Methods 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 description 1
- 229910052721 tungsten Inorganic materials 0.000 description 1
- 239000010937 tungsten Substances 0.000 description 1
- 230000009452 underexpressoin Effects 0.000 description 1
- 101150076562 virB gene Proteins 0.000 description 1
- 101150033532 virG gene Proteins 0.000 description 1
Classifications
-
- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
-
- 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
- C12N15/8255—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 lignin biosynthesis
-
- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
-
- 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
- C12N9/93—Ligases (6)
-
- C—CHEMISTRY; METALLURGY
- 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
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- 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 identification of genes associated with QTL loci of quantity of lignin and digestibility of corn.
- phenotypic traits show continuous variation in populations, which can be quantified. Examples of such traits include the number of branches, precocity, resistance to insects, percentage of protein in the grain, etc. It is recognized that this continuous distribution can be explained by supposing that several segregated loci influence the variation of the character, to which environmental effects can also contribute (de Vienne et al, 1998). Since the early 1980s, it has been customary to call these loci, in English as in French, QTL (for “Quantitative Trait Loci”). Differences in effects between wild alleles are thought to be responsible for the variation in quantitative traits (Helentjaris 1987, Beavis et al. 1991).
- the authors of the present invention are interested, among these quantitative characters, in the digestibility of corn.
- the nutritive quality of the corn of ensiiage depends, in addition to the stage of harvest, (evaluated by the percentage of dry matter which must be included between 30 and 35%), the respective quantities of grains and stems and digestibility specific to each of these two parts.
- the digestibility value of corn is difficult to assess.
- the digestibility of the stem mainly depends on the digestibility of the cell walls.
- the cell walls consist mainly of cellulose, hemicellulose and lignin.
- Lignin unlike the other constituents, is not digested by polygastric animals and is therefore considered limiting for the digestibility value of forages. Correlations between quantity of iignin and digestibility have been published, but the results are contradictory. A negative correlation is generally observed when different stages of maturity are compared, while the correlation is less obvious when a single stage of maturity is considered (Jung and Allen, 1995). This shows that lignin only partially explains the overall variation in digestibility.
- marker locus is meant a locus identified by a polymorphic marker which provides information on the genotype at this locus and in part at neighboring loci due to the genetic link.
- markers include RFLP (“restriction fragment length polymorphism”), RAPD (“random-amplified polymorphic DNA”), AFLP (“amplification fragment length polymorphism” or SSR (“simple sequence repeats”)) markers.
- segregated offspring is meant a population of genetically heterogeneous plants with the same genetic derivation and therefore being related.
- segregated progeny include populations obtained by backcrossing, recombinant lines or populations of F2 lines.
- step (ii) above were carried out by near infrared spectrometry (or NIRS in English).
- a monochromator type device (NIRS System 5000 - FOSS) was used. Wall and lignin contents (percentage relative to walls) and a measure of enzymatic digestibility of the walls according to Aufrere (Avemrere and Demarquilly, 1989) were thus predicted.
- the correlation between the genotype of the marker loci and the digestibility, which allows the localization of the digestibility QTLs as mentioned in step (iii) above, can be carried out by biometric methods known to those skilled in the art (from Vienna , 1998). These biometric methods can be based on marker-by-marker analyzes (Tanskley et al, 1982) or by taking two or more markers together (Landen and Botstein, 1989). By means of this method, described more precisely in the examples below, the authors of the present invention have succeeded in identifying several chromosomal regions as being QTL loci of corn digestibility.
- the present invention more particularly relates to a chromosomal region identified as being a QTL locus of corn digestibility, characterized in that it is delimited by the markers UMC67 and UMC 128 on the chromosome 1 of corn, as indicated in FIG. 1 .
- markers are known to those skilled in the art, and are for example accessible via the "Maize Genome Database” of the University of Missouri (http: //www.agron. Missouri.edu).
- this chromosomal region is around 15% of the phenotypic variance of the lignin content, and around 15% also for enzymatic digestibility.
- the chromosomal region of interest is more particularly delimited by the markers UMC 67, or preferably UMC 58 at one end and by the marker UMC 128 at the other end.
- this chromosomal region identified as being a QTL locus of corn digestibility included the 4CL2 gene coding for enzyme 4-coumarate CoA ligase 2 and the CCR gene coding for the enzyme Cynnamoyl CoA reductase, located between the markers UMC 58 and UMC 128.
- markers can in particular be used to prepare primers for amplification reactions of PCR type (polymerase chain reaction), applied to corn DNA or to prepare probes for Southern hybridizations.
- PCR type polymerase chain reaction
- the subject of the invention is therefore a chromosomal region identified as being a QTL locus of corn digestibility, characterized in that it is between the markers UMC 67 and UMC 128 on the chromosome 1 of corn and that it comprises the gene 4CL2 coding for the enzyme 4-coumarate CoA ligase 2 and the CCR gene coding for the enzyme Cynnamoyl CoA reductase.
- the attached sequence SEQ ID No. 1 corresponds to a full length cDNA coding for corn 4CL2.
- Patent application US 866 791 also describes a recombinant DNA coding for the corn CCR enzyme, also described in the article by Civardi et.al, 1998 and accessible on the GenBank database (access number Y13734 ).
- the authors of the present invention have also highlighted another chromosomal region, identified as being a QTL locus of corn digestibility. This region is characterized in that it is delimited by the markers bnlg 1046 and bnlg 609 on the chromosome 5 of maize, as indicated in FIG. 2.
- the chromosomal region of interest is more particularly delimited by the UMC marker 27a at one end and by the bnl marker 7.71 at the other end.
- this chromosomal region identified as being a QTL locus of corn digestibility included the 4CL1 gene coding for enzyme 4-coumarate CoA ligase 1, the CAD gene coding for the enzyme cinnamyl alcohol dehydrogenase and the gene F5H coding for the enzyme ferulate 5-hydroxylase located between the markers UMC 27a and bnl 7.71.
- markers can in particular be used to prepare primers for amplification reactions of PCR type (polymerase chain reaction), applied to maize DNA or to prepare probes for hybridizations (Southern).
- the invention therefore relates to a chromosomal region identified as being a QTL locus of corn digestibility, characterized in that it is included between the markers bnlg 1046 and bnlg 609 on chromosome 5 of corn and that it comprises the 4CL1 gene coding for the enzyme 4-coumarate CoA ligase 1, the CAD gene coding for the enzyme cinnamyl alcohol dehydrogenase, and the F5H gene coding for a ferulate-5-hydroxylase.
- the full-length cDNA sequence encoding the enzyme 4CL1 is presented in the attached sequence list, SEQ ID No. 2.
- the CAD enzyme and its gene are described in Civardi et al, 1998 and on the Ge ⁇ bank database (Access No. Y13733)
- the annexed sequence SEQ ID No. 3 corresponds to the cDNA coding for corn F5H.
- Nucleic probes or primers allowing amplification by PCR can be constructed from all or part of the chromosomal regions described above or bordering them. They may in particular be probes obtained from the sequences coding for the enzymes CCR, 4CL2, CAD and / or 4CL1 and F5H.
- Marker-assisted selection programs can be implemented using these probes as markers, so as to introgress chromosomal segments in varieties of corn to increase the digestibility value of these varieties.
- the present invention therefore relates to the use of at least one probe or a nucleic primer obtained from all or part of the chromosomal regions as defined above or bordering them, for monitoring the introgression of the QTL of corn digestibility, in a marker-assisted corn selection program.
- these probes can be labeled according to conventional techniques known to a person skilled in the art, for example by a radioactive isotope and used to locate the digestibility QTLs in maize using the Southern technique for example.
- at least one labeled probe is brought into contact with the genomic DNA of a maize, previously digested with restriction enzymes, under appropriate hybridization conditions, then the size of the restriction fragment which hybridizes is determined. with the probe.
- Primers constructed from all or part of the chromosomal regions described above or bordering these regions can also be used to characterize these regions, according to conventional PCR type techniques (using two flanking primers), or quantitative PCR (using two flanking primers and a primer specific for the favorable allele), or even using a technique not using PCR, such as that described and marketed by Third Wave Technology ( Madison, Wl 53719.1256, USA), known as Invader TM.
- the present invention also relates to a method for determining a correlation between the haplotype of the chromosomal regions identified as being a QTL locus of digestibility of a corn and its digestibility value, comprising: a) the haplotyping of all or part of the chromosomal region identified as being a QTL locus of digestibility, this haplotyping step being carried out on individuals belonging to the descent of maize on which the chromosomal regions as defined above have been identified as being a QTL locus of digestibility But ; and / or b) the haplotyping of all or part of the chromosomal region of the digestibility locus QTL as defined above, this haplotyping step being carried out on individuals who are not related to said individuals of step (a) ; c) determining the digestibility value of all of said individuals; d) establishing a correlation between the digestibility value and haplotype profiles obtained.
- Haplotyping consists in searching for assortments of alleles with the different markers of the QTL locus of digestibility.
- Haplotyping conventionally comprises the detection of haplotypes by sequencing all or part of the chromosomal regions defined above and the typing or identification of the haplotype at the positions of sequences identified as polymorphic, by means of nucleotic probes or primers obtained. from all or part of one of the chromosomal regions defined above, using conventional hybridization or amplification techniques.
- the populations subjected to a haplotyping can be made up of individuals descended from a cross used to study the QTL locus, for which alleles favorable to high digestibility are known. They may also be unrelated individuals, that is to say individuals who do not have common direct ancestors.
- a correlation between the haplotype and the digestibility value is established by dividing the population according to the haplotypes present. A correlation or association is observed if the means for the digestibility values vary significantly between the populations sorted according to molecular polymorphism.
- the present invention therefore also relates to a method for predictive determination of the digestibility value of a corn, comprising: a) haplotyping of all or part of the chromosomal region identified as being a QTL locus of digestibility; b) the predictive determination of the digestibility value of said maize, by means of the correlation established previously.
- the selection of corn with high digestibility is then advantageous, and can be carried out by a method comprising: the haplotyping of all or part of the chromosomal region identified as being a QTL locus of digestibility, the selection among these individuals of plants which have a predictive value of high digestibility determined by the method described above.
- This method interestingly complements the methods of selection of corn with a high degree of digestibility, in which one implements: the genotyping of individuals by means of probes or nucleic primers obtained from all or part of a chromosomal region as defined above or bordering it; the selection among these individuals of plants which include a high frequency of favorable alleles associated with digestibility.
- All of these methods allow the determination of the digestibility value of any individuals, not related to the genetic material used for the research of QTL.
- These selection methods allow more particularly the screening of corn representing genetic resources different from those usually used in breeding, and through this the selection of new lines or populations with a high degree of digestibility. They also make it possible to obtain improved corn plants, with a high degree of digestibility, by transfer (in the form of introgression in particular) of the alleles or haplotypes linked to a desired digestibility.
- the invention further relates to a method for obtaining genetically transformed maize having a high digestibility, said method comprising:
- the present invention further extends to a nucleic acid encoding the enzyme 4-coumarate CoA ligase 2 (4CL2), comprising the nucleotide sequence SEQ ID No. 1.
- the invention also covers a nucleic acid coding for the enzyme 4-coumarate CoA ligase (4CL1), comprising the sequence SEQ ID No. 2.
- the subject of the invention is also a nucleic acid coding for the enzyme ferulate-5-hydroxylase (F5H), comprising the sequence SEQ ID No. 3.
- the invention also relates to a vector comprising at least one of the nucleic acids mentioned above, in association with operative sequences allowing its expression. Also included in the invention is a method for obtaining a genetically modified corn, comprising the transformation of a corn plant cell with this nucleic acid or this vector and the regeneration of a transgenic plant from the cell thus transformed. The invention finally relates to a transgenic corn plant or part of this plant, such as seed, leaf, cell, or the like, capable of being obtained by said method.
- FIG. 1 represents the consensus map of the SSR markers of chromosome 1 of maize, accessible on the Internet site (http://www.aqron.missouri.edu/cqi-bin/svbqw mdb / mdb3 / Map / 145822).
- FIG. 2 represents the consensus of the SSR markers of chromosome 5 of corn, accessible on the Internet site (http://www.aqron.missouri.edu/cqi-bin/sybqw mdb / mdb3 / Map / 145826).
- A. Measuring digestibility The plant material used consists of two populations of F2 maize (220 and 140 individuals respectively) obtained by crossing between horny lines for one and toothed lines for the other. The parental lines specific to the same crossing were chosen for their difference in wall digestibility and wall content. The F3 families were evaluated by NIR (“Near Infrared Reflectance”) on two culture sites for different parameters of digestibility (wall, lignin content, and enzymatic digestibility of total organic matter).
- the link between the genotype of the marker loci and the quantitative digestibility variables was carried out by the method based on a QTL search algorithm by "interval mapping" (Mapmaker / QTL software version 1.1, Lincoln et al, 1993). A QTL was declared significant when the Lod score was greater than 2.5. A confidence interval was defined for each QTL by taking for each end the maximum Lod Score - 1.5.
- QTLs were obtained in particular on chromosomes 1 and 5.
- the determination coefficients (R 2 ) given by the mapmaker / QTL software are 20% and 10% respectively of the total phenotypic variance.
- R 2 determination coefficients
- the QTL obtained on chromosome 5 has a confidence interval between the markers bnlg 1046 and bnlg 609. This QTL is found for the two culture sites of one of the two populations with a Lod score of 3.1 and 2.8 and an R 2 of 12% and 10% respectively depending on the place of culture.
- Example 2
- the cDNAs corresponding to these genes are mapped by hybridization on blots of DNA from the population of segregated individuals, digested by restriction enzymes.
- the genetic maps of these populations are saturated with at least one marker on average every 4 cM.
- Mapmaker / Exp software version 3.0 is used to map cDNAs relative to the anchor markers of these genetic maps.
- the protocol is similar to that followed for the co-localization of the 4CL2 and CCR genes.
- the region where CAD, 4CL1 and F5H are mapped is located on chromosome 5 between the markers UMC 27a and bnl 7.71.
- the genetic markers bordering the chromosomal region or included in the chromosomal region characterized according to the invention can thus be used in a program of selection assisted by markers to predict the genotype at the locus of the trait of character by virtue of the link between the locus genetic marker and the locus QTL of corn digestibility on the one hand and of the co -location highlighted between this QTL locus and the genes coding for enzymes involved in the biosynthesis of lignin on the other hand. It is therefore also possible to use as markers, sequences included in these chromosomal regions comprising all or part of the sequences 4CL1 and 2, CCR, CAD, F5H.
- the effect on the digestibility of the QTL allele must be evaluated in relation to the alleles of the molecular markers of the chromosomal zone comprising one of the two QTL described above.
- a population of varieties of corn or hybrids can be genotyped using a set of markers previously mapped on one of the chromosomal zones described above.
- each combination of marker alleles is associated with an allele at QTL and an evaluation of the effect of the allele on digestibility can be carried out by analysis of variance of the digestibility value of the varieties using as a factor modality the allele to QTL.
- the other possibility for assessing the effect of alleles at QTL is the crossing approach: - either by crossing between two lines or varieties; - either by using a series of related or diallel crosses.
- the principle is however the same for these two types of crosses since the effect of the allele at QTL is evaluated by comparison of the digestibility value of the descendants of the crosses.
- two varieties (or two individuals from descendants), respectively having an allele favorable to a good digestibility value for the QTL of chromosome 1 and for the QTL of chromosome 5 are crossed in order to obtain an individual from the offspring of this new cross which contains these two alleles.
- Molecular markers belonging to these two chromosomal zones are then used to identify this. individual.
- This individual can then be fixed by successive self-fertilization, making sure not to lose the two alleles thanks to the molecular markers.
- the two alleles can also be introgressed in a line or variety presenting a good agronomic value, and this introgression is followed by molecular markers bordering the zone of QTLs which it is desired to introgress.
- Target cells are undifferentiated cells in rapid divisions which have retained the ability to regenerate whole plants. This type of cell makes up the embryogenic callus (called type II) of corn. These calluses are obtained from immature Hill genotype embryos according to the method and on the media described by Armstrong (Maize Handbook; 1994 M. Freeling, V. Walbot Eds; pp.665-671).
- the callus boxes thus bombarded are then sealed using Scellofrais R and then grown in the dark at 27 ° C.
- the first subculture takes place 24 hours later, then every two weeks for 3 months on medium identical to the initiation medium added with a selective agent.
- Calls are obtained after 3 months or sometimes earlier, calluses whose growth is not inhibited by the selective agent, usually and mainly composed of cells resulting from the division of a cell having integrated into its genetic heritage one or more copies of the selection gene.
- the frequency of obtaining such calluses is approximately 0.8 cal per bombarded box.
- Agrobacterium tumefaciens uses Agrobacterium tumefaciens, according to the protocol described by Ishida et al (1996), in particular from immature embryos 10 days after fertilization. All the media used are referenced in the cited reference.
- the transformation begins with a co-culture phase where the immature embryos of the corn plants are brought into contact for at least 5 minutes with Agrobacterium tumefaciens LBA 4404 containing the superbinary vectors.
- the superbinary plasmid is the result of homologous recombination between an intermediate vector carrying the T-DNA containing the gene of interest (in this case 4CL1, 4CL2, CAD, CCR and / or F5H), and the vector pSB 1 from Japan Tobacco (EP 672 752) which contains: the virB and virG genes of the plasmid pTiBo542 present in the supervirulent strain A281 of Agrobacterium tumefaciens (ATCC 37349) and a homologous region found in the intermediate vector allowing this homologous recombination.
- the embryos are then placed on LSAs medium for 3 days in the dark and at 25 ° C.
- the second selection step is carried out by transfer of the embryos which have developed on LSD5 medium, on LSD10 medium (phosphinotricin at 10 mg / l) in the presence of cefotaxime, for 3 weeks under the same conditions as above.
- the third selection step consists in excising the type I calluses (fragments of 1 to 2 mm) and transferring them 3 weeks in the dark and at 25 ° C to LSD 10 medium in the presence of cefotaxime.
- the regeneration of the seedlings is carried out by excising the type I calluses which have proliferated and by transferring them to LSZ medium in the presence of phosphinotricin at 5 mg / l and cefotaxime for 2 weeks at 22 ° C. and under continuous light.
- the regenerated seedlings are transferred to RM + G2 medium containing 1,00 mg / l of Augmentin for 2 weeks at 22 ° C. and under continuous illumination for the development stage.
- the plants obtained are then transferred to the phytotron for their acclimatization.
- the invention can be carried out using as starting plant material a population resulting from the Symphony® hybrid.
- Mapmaker / EXP version 3.0 a tutorial and reference manual.
- - Lincoln ES et al Mapping genes controlling quantitative traits using Mapmaker / QTL version 1.1: a tutorial and reference manual.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Botany (AREA)
- Mycology (AREA)
- Cell Biology (AREA)
- Nutrition Science (AREA)
- Immunology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention concerns the identification and co-localisation between two QTL corn digestibility loci and genes involved in the synthesis of lignin, and the uses thereof in methods for producing digestible corn.
Description
Identification de gènes associés à un locus QTL de digestibilité du maïs Identification of genes associated with a QTL locus of corn digestibility
La présente invention concerne l'identification de gènes associés à des loci QTL de quantité de lignine et de digestibilité du maïs.The present invention relates to the identification of genes associated with QTL loci of quantity of lignin and digestibility of corn.
De nombreux caractères phénotypiques manifestent une variation continue dans les populations, variation qui peut être quantifiée. Parmi de tels caractères, on peut citer par exemple le nombre de ramifications, la précocité, la résistance à des insectes, le pourcentage de protéines dans le grain, etc. Il est admis que cette distribution continue peut s'expliquer en supposant que plusieurs locus en ségrégation influencent la variation du caractère, à laquelle des effets de milieu peuvent également contribuer (de Vienne et al, 1998). Depuis le début des années 80, on a coutume d'appeler ces locus, en anglais comme en français, des QTL (pour « Quantitative Trait Loci »). Des différences d'effets entre alleles sauvages seraient responsables de la variation des caractères quantitatifs ( Helentjaris 1987, Beavis et al. 1991 ).Many phenotypic traits show continuous variation in populations, which can be quantified. Examples of such traits include the number of branches, precocity, resistance to insects, percentage of protein in the grain, etc. It is recognized that this continuous distribution can be explained by supposing that several segregated loci influence the variation of the character, to which environmental effects can also contribute (de Vienne et al, 1998). Since the early 1980s, it has been customary to call these loci, in English as in French, QTL (for “Quantitative Trait Loci”). Differences in effects between wild alleles are thought to be responsible for the variation in quantitative traits (Helentjaris 1987, Beavis et al. 1991).
Les auteurs de la présente invention se sont intéressés, parmi ces caractères quantitatifs, à la digestibilité du maïs. La qualité nutritive des maïs d'ensiiage dépend, en plus du stade de récolte, (évalué par le pourcentage de matière sèche qui doit être compris entre 30 et 35%), des quantités respectives des grains et des tiges et des digestibilité propre à chacune de ces deux parties.The authors of the present invention are interested, among these quantitative characters, in the digestibility of corn. The nutritive quality of the corn of ensiiage depends, in addition to the stage of harvest, (evaluated by the percentage of dry matter which must be included between 30 and 35%), the respective quantities of grains and stems and digestibility specific to each of these two parts.
La valeur de digestibilité du maïs est difficile à évaluer. La digestibilité de la tige dépend principalement de la digestibilité des parois des cellules. Les parois des cellules sont constituées principalement de cellulose, d'hémicellulose et de lignine. La lignine contrairement aux autres constituants n'est pas digérée par les animaux polygastriques et elle est donc considérée limitante pour la valeur de digestibilité des fourrages. Des corrélations entre quantité de iignine et digestibilité ont été publiées, mais les résultats sont contradictoires. Une corrélation négative est généralement observée lorsque différents stades de maturité sont comparés alors que la corrélation est moins évidente lorsqu'un seul stade de maturité est considéré (Jung and Allen, 1995).
Ceci témoigne du fait que la lignine n'explique qu'en partie la variation globale de la digestibilité.The digestibility value of corn is difficult to assess. The digestibility of the stem mainly depends on the digestibility of the cell walls. The cell walls consist mainly of cellulose, hemicellulose and lignin. Lignin, unlike the other constituents, is not digested by polygastric animals and is therefore considered limiting for the digestibility value of forages. Correlations between quantity of iignin and digestibility have been published, but the results are contradictory. A negative correlation is generally observed when different stages of maturity are compared, while the correlation is less obvious when a single stage of maturity is considered (Jung and Allen, 1995). This shows that lignin only partially explains the overall variation in digestibility.
A l'origine, les mesures de digestibilité du maïs ensilage sont réalisées en mesurant le gain énergétique de ruminants nourris à partir de cet ensilage. Ces tests in vivo sont cependant coûteux et nécessitent de grandes quantités de matériel végétal. Aussi, des méthodes indirectes ont donc été développées : - mesure de digestibilité in vitro dans du jus de rumen (Menke et al, 1979) ; - mesure de digestibilité in vitro enzymatique (Aufrere and Demarquilly, 1989). Des mesures chimiques sont également utilisées pour quantifier les constituants de paroi (Van Soest, 1963). Une autre méthode d'évaluation de la digestibilité passe par l'utilisation de spectrométrie dans le proche infra-rouge (Ronsin et Femenias, 1990). Cette technique permet de prédire différents paramètres de digestibilité ou de quantité de constituants de paroi. Les auteurs de la présente invention sont maintenant parvenus à établir une co-localisation entre QTL de digestibilité du maïs et certains gènes, connus comme étant impliqués dans les voies de synthèse de la lignine, co- localisation susceptible d'être mise à profit notamment pour produire des maïs plus digestibles.Originally, the measures of digestibility of silage corn are carried out by measuring the energy gain of ruminants fed from this silage. These in vivo tests are however expensive and require large amounts of plant material. Also, indirect methods have therefore been developed: - measurement of digestibility in vitro in rumen juice (Menke et al, 1979); - measurement of enzymatic in vitro digestibility (Aufrere and Demarquilly, 1989). Chemical measurements are also used to quantify the wall constituents (Van Soest, 1963). Another method of assessing digestibility involves the use of near infrared spectrometry (Ronsin and Femenias, 1990). This technique makes it possible to predict different parameters of digestibility or quantity of wall constituents. The authors of the present invention have now managed to establish a co-localization between QTL of corn digestibility and certain genes, known to be involved in the lignin synthesis pathways, co-localization which can be exploited in particular for produce more digestible corn.
Pour cela, ils ont mis en oeuvre une méthode comprenant : i) la détermination, pour plusieurs individus d'une descendance de maïs en ségrégation, du génotype d'un ensemble de locus marqueurs distribués tout le long du génome ; ii) la mesure de la digestibilité pour chacun des individus étudiés ; iii) la corrélation entre le génotype des locus marqueurs et la digestibilité, permettant la localisation des QTL de digestibilité ; iv) la corrélation entre le QTL de digestibilité ainsi localisé et au moins un gène, ledit gène ayant été préalablement cartographie, sur les mêmes individus ou d'autres individus de la même espèce, dans la région desdits locus marqueurs corrélés audit QTL de digestibilité.
Par « locus marqueur», on entend un locus identifié par un marqueur polymorphe qui renseigne sur le génotype à ce locus et en partie aux locus voisins du fait de la liaison génétique. Parmi les marqueurs courants, on peut citer les marqueurs RFLP (« restriction fragment length polymorphism ») RAPD (« random-amplified polymorphic DNA »), AFLP(« amplification fragment length polymorphism » ou SSR (« simple séquence repeats »).For this, they implemented a method comprising: i) the determination, for several individuals of a descent of maize in segregation, of the genotype of a set of marker locus distributed throughout the genome; ii) measurement of digestibility for each of the individuals studied; iii) the correlation between the genotype of marker loci and digestibility, allowing the localization of digestibility QTLs; iv) the correlation between the digestibility QTL thus located and at least one gene, said gene having been previously mapped, on the same individuals or other individuals of the same species, in the region of said marker loci correlated to said digestibility QTL. By “marker locus” is meant a locus identified by a polymorphic marker which provides information on the genotype at this locus and in part at neighboring loci due to the genetic link. Common markers include RFLP (“restriction fragment length polymorphism”), RAPD (“random-amplified polymorphic DNA”), AFLP (“amplification fragment length polymorphism” or SSR (“simple sequence repeats”)) markers.
Par « descendance en ségrégation », on entend une population de plantes génétiquement hétérogènes de même dérivation génétique et étant par conséquent apparentées. Des exemples de descendance en ségrégation incluent des populations obtenues par rétrocroisement, des lignées recombinantes ou des populations de lignées F2.By “segregated offspring” is meant a population of genetically heterogeneous plants with the same genetic derivation and therefore being related. Examples of segregated progeny include populations obtained by backcrossing, recombinant lines or populations of F2 lines.
Les mesures de digestibilité telles que mentionnées à l'étape (ii) ci-dessus ont été réalisées par spectrométrie dans le proche infrarouge (ou NIRS en anglais). Un appareil de type monochromateur (NIRS System 5000 - FOSS) a été utilisé. Les teneurs en paroi et en lignine (pourcentage par rapport aux parois) et une mesure de digestibilité enzymatique des parois selon Aufrere (Aufrere et Demarquilly, 1989) ont ainsi été prédites.The digestibility measurements as mentioned in step (ii) above were carried out by near infrared spectrometry (or NIRS in English). A monochromator type device (NIRS System 5000 - FOSS) was used. Wall and lignin contents (percentage relative to walls) and a measure of enzymatic digestibility of the walls according to Aufrere (Aufrere and Demarquilly, 1989) were thus predicted.
La corrélation entre le génotype des locus marqueurs et la digestibilité, qui permet la localisation des QTL de digestibilité comme mentionné à l'étape (iii) ci-dessus, peut être effectuée par des méthodes biométriques connues de l'homme du métier (de Vienne, 1998). Ces méthodes biométriques peuvent s'appuyer sur des analyses marqueur par marqueur (Tanskley et al, 1982) ou en prenant en compte conjointement deux ou plusieurs marqueurs (Landen et Botstein, 1989). Au moyen de cette méthode, décrite plus précisément dans les exemples ci-après, les auteurs de la présente invention sont parvenus à identifier plusieurs régions chromosomiques comme étant des loci QTL de digestibilité du maïs.The correlation between the genotype of the marker loci and the digestibility, which allows the localization of the digestibility QTLs as mentioned in step (iii) above, can be carried out by biometric methods known to those skilled in the art (from Vienna , 1998). These biometric methods can be based on marker-by-marker analyzes (Tanskley et al, 1982) or by taking two or more markers together (Landen and Botstein, 1989). By means of this method, described more precisely in the examples below, the authors of the present invention have succeeded in identifying several chromosomal regions as being QTL loci of corn digestibility.
La présente invention a plus particulièrement pour objet une région chromosomique identifiée comme étant un locus QTL de digestibilité du maïs, caractérisée en ce qu'elle est délimitée par les marqueurs UMC67 et UMC 128 sur le chromosome 1 du maïs, comme indiqué à la figure 1.
Ces marqueurs sont connus de l'homme du métier, et sont par exemple accessibles via la "Maize Génome Database" de l'Université du Missouri (http : //www.agron. missouri.edu).The present invention more particularly relates to a chromosomal region identified as being a QTL locus of corn digestibility, characterized in that it is delimited by the markers UMC67 and UMC 128 on the chromosome 1 of corn, as indicated in FIG. 1 . These markers are known to those skilled in the art, and are for example accessible via the "Maize Genome Database" of the University of Missouri (http: //www.agron. Missouri.edu).
La contribution de cette région chromosomique est de l'ordre de 15 % de la variance phénotypique de la teneur en lignine, et de l'ordre de 15 % également pour la digestibilité enzymatique.The contribution of this chromosomal region is around 15% of the phenotypic variance of the lignin content, and around 15% also for enzymatic digestibility.
La région chromosomique d'intérêt est plus particulièrement délimitée par les marqueurs UMC 67, ou de préférence UMC 58 à une extrémité et par le marqueur UMC 128 à l'autre extrémité. En application de l'étape (iv) de la méthode décrite ci-dessus, les auteurs de la présente invention ont mis en évidence que cette région chromosomique identifiée comme étant un locus QTL de digestibilité du maïs, comprenait le gène 4CL2 codant pour l'enzyme 4-coumarate CoA ligase 2 et le gène CCR codant pour l'enzyme Cynnamoyl CoA réductase, localisés entre les marqueurs UMC 58 et UMC 128.The chromosomal region of interest is more particularly delimited by the markers UMC 67, or preferably UMC 58 at one end and by the marker UMC 128 at the other end. In application of step (iv) of the method described above, the authors of the present invention have demonstrated that this chromosomal region identified as being a QTL locus of corn digestibility, included the 4CL2 gene coding for enzyme 4-coumarate CoA ligase 2 and the CCR gene coding for the enzyme Cynnamoyl CoA reductase, located between the markers UMC 58 and UMC 128.
Ces marqueurs peuvent notamment servir pour préparer des amorces pour des réactions d'amplification de type PCR (réaction en chaîne par polymérase), appliquées à de l'ADN de maïs ou pour préparer des sondes pour des hybridations Southern.These markers can in particular be used to prepare primers for amplification reactions of PCR type (polymerase chain reaction), applied to corn DNA or to prepare probes for Southern hybridizations.
L'invention a donc pour objet une région chromosomique identifiée comme étant un locus QTL de digestibilité du maïs, caractérisée en ce qu'elle est comprise entre les marqueurs UMC 67 et UMC 128 sur le chromosome 1 du maïs et qu'elle comprend le gène 4CL2 codant pour l'enzyme 4-coumarate CoA ligase 2 et le gène CCR codant pour l'enzyme Cynnamoyl CoA réductase.The subject of the invention is therefore a chromosomal region identified as being a QTL locus of corn digestibility, characterized in that it is between the markers UMC 67 and UMC 128 on the chromosome 1 of corn and that it comprises the gene 4CL2 coding for the enzyme 4-coumarate CoA ligase 2 and the CCR gene coding for the enzyme Cynnamoyl CoA reductase.
La séquence SEQ ID n°1 annexée correspond à un ADNc de pleine longueur codant pour la 4CL2 de maïs.The attached sequence SEQ ID No. 1 corresponds to a full length cDNA coding for corn 4CL2.
La demande de brevet US 866 791 décrit par ailleurs un ADN recombinant codant pour l'enzyme CCR de maïs, également décrit dans l'article de Civardi et.al, 1998 et accessible sur la base de données GenBank (n° d'accès Y13734).
Les auteurs de la présente invention ont également mis en évidence une autre région chromosomique, identifiée comme étant un locus QTL de digestibilité du maïs. Cette région est caractérisée en ce qu'elle est délimitée par les marqueurs bnlg 1046 et bnlg 609 sur le chromosome 5 du maïs, comme indiqué à la figure 2.Patent application US 866 791 also describes a recombinant DNA coding for the corn CCR enzyme, also described in the article by Civardi et.al, 1998 and accessible on the GenBank database (access number Y13734 ). The authors of the present invention have also highlighted another chromosomal region, identified as being a QTL locus of corn digestibility. This region is characterized in that it is delimited by the markers bnlg 1046 and bnlg 609 on the chromosome 5 of maize, as indicated in FIG. 2.
Ces marqueurs sont connus de l'homme du métier, et sont par exemple donnés par le "Maize Génome Database" de l'Université du Missouri.These markers are known to those skilled in the art, and are for example given by the "Maize Genome Database" of the University of Missouri.
La contribution de cette région chromosomique par rapport à la teneur en lignine est de l'ordre de 10 % de la variance phénotypique.The contribution of this chromosomal region relative to the lignin content is of the order of 10% of the phenotypic variance.
La région chromosomique d'intérêt est plus particulièrement délimitée par le marqueur UMC 27a à une extrémité et par le marqueur bnl 7.71 à l'autre extrémité.The chromosomal region of interest is more particularly delimited by the UMC marker 27a at one end and by the bnl marker 7.71 at the other end.
En application de l'étape (iv) de la méthode décrite ci-dessus, les auteurs de la présente invention ont mis en évidence que cette région chromosomique identifiée comme étant un locus QTL de digestibilité du maïs, comprenait le gène 4CL1 codant pour l'enzyme 4-coumarate CoA ligase 1 , le gène CAD codant pour l'enzyme cinnamyl alcool deshydrogénase et le gène F5H codant pour l'enzyme ferulate 5-hydroxylase localisés entre les marqueurs UMC 27a et bnl 7.71.In application of step (iv) of the method described above, the authors of the present invention have demonstrated that this chromosomal region identified as being a QTL locus of corn digestibility, included the 4CL1 gene coding for enzyme 4-coumarate CoA ligase 1, the CAD gene coding for the enzyme cinnamyl alcohol dehydrogenase and the gene F5H coding for the enzyme ferulate 5-hydroxylase located between the markers UMC 27a and bnl 7.71.
Ces marqueurs peuvent notamment servir pour préparer des amorces pour des réactions d'amplification de type PCR (réaction en chaîne par polymérase), appliquées à de l'ADN de maïs ou pour préparer des sondes pour des hybridations (Southern).These markers can in particular be used to prepare primers for amplification reactions of PCR type (polymerase chain reaction), applied to maize DNA or to prepare probes for hybridizations (Southern).
?5 L'invention a donc pour objet une région chromosomique identifiée comme étant un locus QTL de digestibilité du maïs, caractérisée en ce qu'elle est comprise entre les marqueurs bnlg 1046 et bnlg 609 sur le chromosome 5 du maïs et qu'elle comprend le gène 4CL1 codant pour l'enzyme 4-coumarate CoA ligase 1 , le gène CAD codant pour l'enzyme 30 cinnamyl alcool deshydrogénase, et le gène F5H codant pour une ferulate-5- hydroxylase.The invention therefore relates to a chromosomal region identified as being a QTL locus of corn digestibility, characterized in that it is included between the markers bnlg 1046 and bnlg 609 on chromosome 5 of corn and that it comprises the 4CL1 gene coding for the enzyme 4-coumarate CoA ligase 1, the CAD gene coding for the enzyme cinnamyl alcohol dehydrogenase, and the F5H gene coding for a ferulate-5-hydroxylase.
La séquence d'ADNc de pleine longueur codant pour l'enzyme 4CL1 est présentée dans la liste de séquences annexée, SEQ ID n° 2.
L'enzyme CAD et son gène sont décrits dans Civardi et al, 1998 et sur la base de données Geπbank (N° d'accès Y13733)The full-length cDNA sequence encoding the enzyme 4CL1 is presented in the attached sequence list, SEQ ID No. 2. The CAD enzyme and its gene are described in Civardi et al, 1998 and on the Geπbank database (Access No. Y13733)
La séquence SEQ ID n° 3 annexée correspond à l'ADNc codant pour la F5H de maïs.The annexed sequence SEQ ID No. 3 corresponds to the cDNA coding for corn F5H.
L'identification de QTL de digestibilité et la mise en évidence des marqueurs et des gènes associés à ces QTL conformément à l'invention ouvre la voie à un certain nombre d'applications.The identification of digestibility QTLs and the demonstration of the markers and genes associated with these QTLs in accordance with the invention opens the way to a certain number of applications.
Des sondes nucléiques ou des amorces permettant une amplification par PCR peuvent être construites à partir de tout ou partie des régions chromosomiques décrites plus haut ou bordant celles-ci. Il peut notamment s'agir de sondes obtenues à partir des séquences codant pour les enzymes CCR, 4CL2, CAD et/ou 4CL1 et F5H.Nucleic probes or primers allowing amplification by PCR can be constructed from all or part of the chromosomal regions described above or bordering them. They may in particular be probes obtained from the sequences coding for the enzymes CCR, 4CL2, CAD and / or 4CL1 and F5H.
Des programmes de sélection assistée par marqueurs peuvent être mis en œuvre en utilisant ces sondes comme marqueurs, de manière à introgresser des segments chromosomiques dans des variétés de maïs pour augmenter la valeur de digestibilité de ces variétés. La présente invention a donc pour objet l'utilisation d'au moins une sonde ou une amorce nucléique obtenue à partir de tout ou partie des régions chromosomiques telles que définies précédemment ou bordant celles-ci, pour le suivi de l'introgression du QTL de digestibilité du maïs, dans un programme de sélection de maïs assistée par marqueurs.Marker-assisted selection programs can be implemented using these probes as markers, so as to introgress chromosomal segments in varieties of corn to increase the digestibility value of these varieties. The present invention therefore relates to the use of at least one probe or a nucleic primer obtained from all or part of the chromosomal regions as defined above or bordering them, for monitoring the introgression of the QTL of corn digestibility, in a marker-assisted corn selection program.
Par exemple ces sondes peuvent être marquées selon les techniques classiques connues de l'homme du métier, par exemple par un isotope radioactif et utilisées pour repérer les QTL de digestibilité chez le maïs grâce à la technique du Southern par exemple. Pour cela, au moins une sonde marquée est mise en contact avec de l'ADN génomique d'un maïs, préalablement digéré par des enzymes de restriction, dans des conditions d'hybridation appropriées, puis on détermine la taille du fragment de restriction qui hybride avec la sonde. Des amorces construites à partir de tout ou partie des régions chromosomiques décrites plus haut ou bordant ces régions peuvent également être utilisées pour caractériser ces régions, selon des techniques de type PCR classique (à l'aide de deux amorces flanquantes), ou
de PCR quantitative (à l'aide de deux amorces flanquantes et d'une amorce spécifique de l'allèle favorable), ou encore au moyen d'une technique n'utilisant pas la PCR, comme celle décrite et commercialisée par Third Wave Technology (Madison, Wl 53719.1256, USA), dénommée Invader™ .For example, these probes can be labeled according to conventional techniques known to a person skilled in the art, for example by a radioactive isotope and used to locate the digestibility QTLs in maize using the Southern technique for example. For this, at least one labeled probe is brought into contact with the genomic DNA of a maize, previously digested with restriction enzymes, under appropriate hybridization conditions, then the size of the restriction fragment which hybridizes is determined. with the probe. Primers constructed from all or part of the chromosomal regions described above or bordering these regions can also be used to characterize these regions, according to conventional PCR type techniques (using two flanking primers), or quantitative PCR (using two flanking primers and a primer specific for the favorable allele), or even using a technique not using PCR, such as that described and marketed by Third Wave Technology ( Madison, Wl 53719.1256, USA), known as Invader ™.
La présente invention a également pour objet une méthode de détermination d'une corrélation entre l'haplotype des régions chromosomiques identifiées comme étant un locus QTL de digestibilité d'un maïs et sa valeur de digestibilité, comprenant : a) l'haplotypage de tout ou partie de la région chromosomique identifiée comme étant un locus QTL de digestibilité, cette étape d'haplotypage étant réalisée sur des individus appartenant à la descendance des maïs sur lesquels les régions chromosomiques telles que définies précédemment ont été identifiées comme étant un locus QTL de digestibilité du maïs ; et/ou b) l'haplotypage de tout ou partie de la région chromosomique du locus QTL de digestibilité telle que définie précédemment, cette étape d'haplotypage étant réalisée sur des individus qui ne sont pas apparentés auxdits individus de l'étape (a) ; c) la détermination de la valeur de digestibilité de l'ensemble desdits individus ; d) l'établissement d'une corrélation entre la valeur de digestibilité et les profils haplotypiques obtenus.The present invention also relates to a method for determining a correlation between the haplotype of the chromosomal regions identified as being a QTL locus of digestibility of a corn and its digestibility value, comprising: a) the haplotyping of all or part of the chromosomal region identified as being a QTL locus of digestibility, this haplotyping step being carried out on individuals belonging to the descent of maize on which the chromosomal regions as defined above have been identified as being a QTL locus of digestibility But ; and / or b) the haplotyping of all or part of the chromosomal region of the digestibility locus QTL as defined above, this haplotyping step being carried out on individuals who are not related to said individuals of step (a) ; c) determining the digestibility value of all of said individuals; d) establishing a correlation between the digestibility value and haplotype profiles obtained.
L'haplotypage consiste à rechercher les assortiments d'allèles aux différents marqueurs du locus QTL de digestibilité. L'haplotypage comprend de manière classique la mise en évidence des haplotypes par le séquençage de tout ou partie des régions chromosomiques définies précédemment et le typage ou identification de l'haplotype aux positions de séquences identifiées comme polymorphes, au moyen de sondes ou amorces nucléotiques obtenues à partir de tout ou partie d'une des régions chromosomiques définies précédemment, à l'aide de techniques classiques d'hybridation ou d'amplification.Haplotyping consists in searching for assortments of alleles with the different markers of the QTL locus of digestibility. Haplotyping conventionally comprises the detection of haplotypes by sequencing all or part of the chromosomal regions defined above and the typing or identification of the haplotype at the positions of sequences identified as polymorphic, by means of nucleotic probes or primers obtained. from all or part of one of the chromosomal regions defined above, using conventional hybridization or amplification techniques.
Les populations soumises à un haplotypage peuvent être constituées d'individus en descendance d'un croisement utilisé pour étudier les
locus QTL, pour lesquels les alleles favorables à une digestibilité élevée sont connus. Il peut en outre s'agir d'individus non apparentés, c'est-à-dire d'individus qui n'ont pas d'ascendants directs communs.The populations subjected to a haplotyping can be made up of individuals descended from a cross used to study the QTL locus, for which alleles favorable to high digestibility are known. They may also be unrelated individuals, that is to say individuals who do not have common direct ancestors.
Une corrélation entre l'haplotype et la valeur de digestibilité est établie en repartissant la population selon les haplotypes en présence. Une corrélation ou association est observée si les moyennes pour les valeurs de digestibilité varient significativement entre les populations triées selon le polymorphisme moléculaire.A correlation between the haplotype and the digestibility value is established by dividing the population according to the haplotypes present. A correlation or association is observed if the means for the digestibility values vary significantly between the populations sorted according to molecular polymorphism.
Une fois que ces polymorphismes moléculaires liés au caractère de digestibilité sont identifiés, une sélection des individus de digestibilité inconnue peut être réalisée, à partir de l'haplotypage du locus QTL de digestibilité.Once these molecular polymorphisms linked to the digestibility character are identified, a selection of individuals of unknown digestibility can be carried out, by haplotyping the QTL locus of digestibility.
La présente invention a donc également pour objet une méthode de détermination prédictive de la valeur de digestibilité d'un maïs, comprenant : a) l'haplotypage de tout ou partie de la région chromosomique identifiée comme étant un locus QTL de digestibilité ; b) la détermination prédictive de la valeur de digestibilité dudit maïs, au moyen de la corrélation établie précédemment.The present invention therefore also relates to a method for predictive determination of the digestibility value of a corn, comprising: a) haplotyping of all or part of the chromosomal region identified as being a QTL locus of digestibility; b) the predictive determination of the digestibility value of said maize, by means of the correlation established previously.
Par suite, la sélection de maïs à digestibilité élevée est alors avantageuse, et peut être effectuée par une méthode comprenant : l'haplotypage de tout ou partie de la région chromosomique identifiée comme étant un locus QTL de digestibilité, la sélection parmi ces individus des plantes qui présentent une valeur prédictive de digestibilité élevée déterminée par la méthode décrite ci-dessus.Consequently, the selection of corn with high digestibility is then advantageous, and can be carried out by a method comprising: the haplotyping of all or part of the chromosomal region identified as being a QTL locus of digestibility, the selection among these individuals of plants which have a predictive value of high digestibility determined by the method described above.
Cette méthode complète de manière intéressante les méthodes de sélection de maïs ayant un degré de digestibilité élevée, dans lesquelles on met en œuvre : le génotypage d'individus au moyen de sondes ou d'amorces nucléiques obtenues à partir de tout ou partie d'une région chromosomique telle que définie précédemment ou bordant celle-ci ;
la sélection parmi ces individus des plantes qui comprennent une fréquence élevée d'allèles favorables associés à la digestibilité.This method interestingly complements the methods of selection of corn with a high degree of digestibility, in which one implements: the genotyping of individuals by means of probes or nucleic primers obtained from all or part of a chromosomal region as defined above or bordering it; the selection among these individuals of plants which include a high frequency of favorable alleles associated with digestibility.
L'ensemble de ces méthodes permet en effet la détermination de la valeur de digestibilité d'individus quelconques, non apparentés au matériel génétique utilisé pour la recherche de QTL. Ces méthodes de sélection permettent plus particulièrement le criblage de maïs représentant des ressources génétiques différentes de celles habituellement utilisées en sélection, et par ce biais la sélection de nouvelles lignées ou populations à degré de digestibilité élevée. Elles permettent également d'obtenir des plantes de maïs améliorées, à degré de digestibilité élevée, par transfert (sous forme d'introgression notamment) des alleles ou haplotypes liés à une digestibilité désirée.All of these methods allow the determination of the digestibility value of any individuals, not related to the genetic material used for the research of QTL. These selection methods allow more particularly the screening of corn representing genetic resources different from those usually used in breeding, and through this the selection of new lines or populations with a high degree of digestibility. They also make it possible to obtain improved corn plants, with a high degree of digestibility, by transfer (in the form of introgression in particular) of the alleles or haplotypes linked to a desired digestibility.
L'invention porte en outre sur une méthode d'obtention de maïs génétiquement transformé ayant une digestibilité élevée, ladite méthode comprenant :The invention further relates to a method for obtaining genetically transformed maize having a high digestibility, said method comprising:
(i) la transformation d'une cellule de plant de maïs avec au moins deux séquences nucleotidiques codant pour des enzymes différentes l'une de l'autre, lesdites séquences étant choisies parmi une séquence d'un allèle favorable à une digestibilité élevée codant pour la CCR, une séquence d'un allèle favorable à une digestibilité élevée codant pour la 4CL2, une séquence d'un allèle favorable à une digestibilité élevée pour la 4CL1 , une séquence d'un allèle favorable à une digestibilité élevée codant pour la CAD, et une séquence d'un allèle favorable à une digestibilité élevée codant pour la F5H, lesdites séquences étant portées par un ou plusieurs acides nucléiques ; et(i) the transformation of a corn plant cell with at least two nucleotide sequences coding for enzymes different from each other, said sequences being chosen from a sequence of an allele favorable to high digestibility coding for CCR, a sequence of an allele favorable to high digestibility coding for 4CL2, a sequence of an allele favorable to high digestibility for 4CL1, a sequence of an allele favorable to high digestibility coding for CAD, and a sequence of an allele favorable to high digestibility coding for F5H, said sequences being carried by one or more nucleic acids; and
(ii) la régénération d'une plante transgénique à partir de la cellule ainsi transformée.(ii) the regeneration of a transgenic plant from the cell thus transformed.
La présente invention s'étend par ailleurs à un acide nucléique codant pour l'enzyme 4-coumarate CoA ligase 2 (4CL2), comprenant ia séquence nucléotidique SEQ ID n°1.
L'invention couvre également un acide nucléique codant pour l'enzyme 4-coumarate CoA ligase (4CL1 ), comprenant la séquence SEQ ID n° 2.The present invention further extends to a nucleic acid encoding the enzyme 4-coumarate CoA ligase 2 (4CL2), comprising the nucleotide sequence SEQ ID No. 1. The invention also covers a nucleic acid coding for the enzyme 4-coumarate CoA ligase (4CL1), comprising the sequence SEQ ID No. 2.
L'invention a en outre pour objet un acide nucléique codant pour l'enzyme ferulate-5-hydroxylase (F5H), comprenant la séquence SEQ ID n°3.The subject of the invention is also a nucleic acid coding for the enzyme ferulate-5-hydroxylase (F5H), comprising the sequence SEQ ID No. 3.
L'invention concerne également un vecteur comprenant l'un au moins des acides nucléiques cités ci-dessus, en association avec des séquences opérantes permettant son expression. Est en outre comprise dans l'invention une méthode d'obtention d'un maïs génétiquement modifié, comprenant la transformation d'une cellule de plant de maïs par cet acide nucléique ou ce vecteur et la régénération d'une plante transgénique à partir de la cellule ainsi transformée. L'invention concerne enfin une plante de maïs transgénique ou partie de cette plante, telle que graine, feuille, cellule, ou autres, susceptible d'être obtenue par ladite méthode.The invention also relates to a vector comprising at least one of the nucleic acids mentioned above, in association with operative sequences allowing its expression. Also included in the invention is a method for obtaining a genetically modified corn, comprising the transformation of a corn plant cell with this nucleic acid or this vector and the regeneration of a transgenic plant from the cell thus transformed. The invention finally relates to a transgenic corn plant or part of this plant, such as seed, leaf, cell, or the like, capable of being obtained by said method.
Les exemples et figures suivants illustrent l'invention sans en limiter la portée d'une quelconque manière.The following examples and figures illustrate the invention without limiting its scope in any way.
LEGENDE DES FIGURES : La figure 1 représente la carte consensus des marqueurs SSR du chromosome 1 du maïs, accessible sur le site de l'Internet (http://www.aqron.missouri.edu/cqi-bin/svbqw mdb/mdb3/Map/145822).LEGEND TO THE FIGURES: FIG. 1 represents the consensus map of the SSR markers of chromosome 1 of maize, accessible on the Internet site (http://www.aqron.missouri.edu/cqi-bin/svbqw mdb / mdb3 / Map / 145822).
La figure 2 représente le consensus des marqueurs SSR du chromosome 5 du maïs, accessible sur le site de l'Internet (http://www.aqron.missouri.edu/cqi-bin/sybqw mdb/mdb3/Map/145826).FIG. 2 represents the consensus of the SSR markers of chromosome 5 of corn, accessible on the Internet site (http://www.aqron.missouri.edu/cqi-bin/sybqw mdb / mdb3 / Map / 145826).
EXEMPLES :EXAMPLES:
Exemple 1 : Localisation des QTL de digestibilité :Example 1: Location of the digestibility QTLs:
A. Mesure de la digestibilité :
Le matériel végétal utilisé se compose de deux populations de maïs F2 (de 220 et 140 individus respectivement) obtenus par croisement entre lignées cornées pour l'une et lignées dentées pour l'autre. Les lignées parentales propres à un même croisement ont été choisies pour leur différence de digestibilité de paroi et de teneur en paroi. Les familles F3 ont été évaluées par NIR (« Near Infrared Réflectance ») sur deux lieux de culture pour différents paramètres de digestibilité (teneur en paroi, en lignine, et digestibilité enzymatique de la matière organique totale).A. Measuring digestibility: The plant material used consists of two populations of F2 maize (220 and 140 individuals respectively) obtained by crossing between horny lines for one and toothed lines for the other. The parental lines specific to the same crossing were chosen for their difference in wall digestibility and wall content. The F3 families were evaluated by NIR (“Near Infrared Reflectance”) on two culture sites for different parameters of digestibility (wall, lignin content, and enzymatic digestibility of total organic matter).
B. Etudes biométriques :B. Biometric studies:
La liaison entre le génotype des locus marqueurs et les variables quantitatives de digestibilité a été réalisée par la méthode basée sur un algorithme de recherche de QTL par "interval mapping" (logiciel Mapmaker/QTL version 1.1 , Lincoln et al, 1993). Un QTL a été déclaré significatif lorsque le Lod score était supérieur à 2.5. Un intervalle de confiance a été défini pour chaque QTL en prenant pour chaque extrémité le Lod Score maximum - 1.5.The link between the genotype of the marker loci and the quantitative digestibility variables was carried out by the method based on a QTL search algorithm by "interval mapping" (Mapmaker / QTL software version 1.1, Lincoln et al, 1993). A QTL was declared significant when the Lod score was greater than 2.5. A confidence interval was defined for each QTL by taking for each end the maximum Lod Score - 1.5.
Après cartographie des familles F3, des QTL ont été obtenus notamment sur les chromosomes 1 et 5. L'intervalle de confiance (=Lod score maximum - 1.5) du QTL sur le chromosome 1 est compris entre les marqueurs UMC 67 et UMC 128 et ce QTL présente pour la teneur en lignine un Lod score de 6.3 pour une population et un lieu de culture et de 2.8 pour l'autre population sur le même lieu de culture. Les coefficients de détermination (R2) donnés par le logiciel mapmaker/ QTL sont de 20 % et 10 % respectivement de la variance phénotypique totale. Avec le même intervalle de confiance un QTL de digestibilité enzymatique de la matière organique est montré pour un lieu de culture et une population. Ce QTL présente un Lod score de 4.5 et un R2 de 13 % de la variance phénotypique totale.After mapping the F3 families, QTLs were obtained in particular on chromosomes 1 and 5. The confidence interval (= maximum Lod score - 1.5) of QTL on chromosome 1 is between the markers UMC 67 and UMC 128 and this QTL presents a Lod score for the lignin content of 6.3 for one population and one place of culture and 2.8 for the other population on the same place of culture. The determination coefficients (R 2 ) given by the mapmaker / QTL software are 20% and 10% respectively of the total phenotypic variance. With the same confidence interval, a QTL of enzymatic digestibility of organic matter is shown for a place of culture and a population. This QTL has a Lod score of 4.5 and an R 2 of 13% of the total phenotypic variance.
Le QTL obtenu sur le chromosome 5 présente un intervalle de confiance compris entre les marqueurs bnlg 1046 et bnlg 609. Ce QTL est retrouvé pour les deux lieux de culture d'une des deux populations avec un Lod score de 3,1 et 2,8 et un R2 de 12% et 10% respectivement suivant le lieu de culture.
Exemple 2 :The QTL obtained on chromosome 5 has a confidence interval between the markers bnlg 1046 and bnlg 609. This QTL is found for the two culture sites of one of the two populations with a Lod score of 3.1 and 2.8 and an R 2 of 12% and 10% respectively depending on the place of culture. Example 2:
Colocalisation des gènes 4CL2 et CCR :Colocation of the 4CL2 and CCR genes:
Deux ADNc codant l'un pour la 4CL2, l'autre pour la CCR ont été cartographiés à partir d'une population en ségrégation, et une distance de 1 ,7Two cDNAs, one coding for 4CL2 and the other for CCR, were mapped from a segregated population, and a distance of 1.7.
% de recombinaison a été observée entre ces deux ADNc (1 individu recombiné sur 60). Cette région est située sur le chromosome 1 entre les marqueurs UMC 58 et UMC 128.% of recombination was observed between these two cDNAs (1 individual recombined out of 60). This region is located on chromosome 1 between the markers UMC 58 and UMC 128.
Les ADNc correspondants à ces gènes sont cartographiés par hybridation sur des transferts ("blots") d'ADN de population d'individus en ségrégation, digéré par des enzymes de restriction. Les cartes génétiques de ces populations sont saturées avec au moins un marqueur en moyenne tous les 4 cM. Le logiciel Mapmaker/Exp version 3.0 est utilisé pour cartographier les ADNc par rapport aux marqueurs ancres de ces cartes génétiques.The cDNAs corresponding to these genes are mapped by hybridization on blots of DNA from the population of segregated individuals, digested by restriction enzymes. The genetic maps of these populations are saturated with at least one marker on average every 4 cM. Mapmaker / Exp software version 3.0 is used to map cDNAs relative to the anchor markers of these genetic maps.
Exemple 3 :Example 3:
Colocalisation des gènes CAD, 4CL1 et F5H :Colocation of the CAD, 4CL1 and F5H genes:
Le protocole est similaire à celui suivi pour la co-localisation des gènes 4CL2 et CCR. La région où se cartographient CAD, 4CL1 et F5H se situe sur le chromosome 5 entre les marqueurs UMC 27a et bnl 7.71.The protocol is similar to that followed for the co-localization of the 4CL2 and CCR genes. The region where CAD, 4CL1 and F5H are mapped is located on chromosome 5 between the markers UMC 27a and bnl 7.71.
Exemple 4 :Example 4:
Obtention de lignées plus digestibles A. Par sélection assistée par marqueurs : De multiples utilisations des marqueurs en sélection sont envisageables : on peut citer l'évaluation et la gestion des ressources génétiques (Song. et al., 1988), le transfert de gènes par rétrocroisement (Young et Tanksley, 1989), la construction de nouveaux génotypes (Lefort- Buson et al., 1990), l'haplotypage et la recherche d'association phénotype- haplotype pour une utilisation plus large de l'information issue de l'étude des QTL (criblage des ressources génétiques).Obtaining more digestible lines A. By selection assisted by markers: Multiple uses of markers in selection are possible: we can cite the evaluation and management of genetic resources (Song. Et al., 1988), gene transfer by backcrossing (Young and Tanksley, 1989), the construction of new genotypes (Lefort-Buson et al., 1990), haplotyping and the search for a phenotype-haplotype association for a wider use of information from the QTL study (screening of genetic resources).
De manière générale, les marqueurs génétiques bordant la région chromosomique ou compris dans la région chromosomique caractérisée selon
l'invention peuvent ainsi être utilisés dans un programme de sélection assistée par marqueurs pour prédire le génotype au locus du trait de caractère en vertu de la liaison entre le locus marqueur génétique et le locus QTL de digestibilité maïs d'une part et de la co-localisation mise en évidence entre ce locus QTL et les gènes codant pour des enzymes impliquées dans la biosynthèse de la lignine d'autre part. On peut donc également utiliser comme marqueurs, des séquences comprises dans ces régions chromosomiques comprenant tout ou partie des séquences 4CL1 et 2, CCR, CAD, F5H.In general, the genetic markers bordering the chromosomal region or included in the chromosomal region characterized according to the invention can thus be used in a program of selection assisted by markers to predict the genotype at the locus of the trait of character by virtue of the link between the locus genetic marker and the locus QTL of corn digestibility on the one hand and of the co -location highlighted between this QTL locus and the genes coding for enzymes involved in the biosynthesis of lignin on the other hand. It is therefore also possible to use as markers, sequences included in these chromosomal regions comprising all or part of the sequences 4CL1 and 2, CCR, CAD, F5H.
Dans une première étape, l'effet sur la digestibilité de l'allèle au QTL doit être évalué en relation avec les alleles des marqueurs moléculaires de la zone chromosomique comprenant un des deux QTL ci-dessus décrits.In a first step, the effect on the digestibility of the QTL allele must be evaluated in relation to the alleles of the molecular markers of the chromosomal zone comprising one of the two QTL described above.
Ainsi une population de variétés de maïs ou d'hybrides peut être génotypée à partir d'un jeu de marqueurs cartographiés au préalable sur une des zones chromosomiques ci-dessus décrites. Pour chaque jeu de marqueurs, chaque combinaison d'allèles de marqueurs est associée à un allèle au QTL et une évaluation de l'effet de l'allèle sur la digestibilité peut être réalisée par analyse de variance de la valeur de digestibilité des variétés en utilisant comme modalité du facteur l'allèle au QTL.Thus a population of varieties of corn or hybrids can be genotyped using a set of markers previously mapped on one of the chromosomal zones described above. For each set of markers, each combination of marker alleles is associated with an allele at QTL and an evaluation of the effect of the allele on digestibility can be carried out by analysis of variance of the digestibility value of the varieties using as a factor modality the allele to QTL.
L'autre possibilité pour évaluer l'effet des alleles au QTL est l'approche par croisement: - soit par croisement entre deux lignées ou variétés; - soit par utilisation d'une série de croisements apparentés ou diallèle. Le principe est toutefois le même pour ces deux types de croisements puisque l'effet de l'allèle au QTL est évalué par comparaison de la valeur de digestibilité des descendants des croisements. Dans une deuxième étape, deux variétés (ou deux individus issus de descendance), ayant respectivement un allèle favorable à une bonne valeur de digestibilité pour le QTL du chromosome 1 et pour le QTL du chromosome 5, sont croisées en vue d'obtenir un individu issu de la descendance de ce nouveau croisement qui contienne ces deux alleles. Des marqueurs moléculaires appartenant à ces deux zones chromosomiques sont alors utilisés pour identifier cet . individu. Cet individu peut alors être fixé par autofécondations successives en s'assurant de ne pas perdre les deux alleles grâce aux marqueurs moléculaires. A partir de cet individu ou à partir des
lignées parentales de cet individu, les deux alleles peuvent également être introgressés dans une lignée ou variété présentant une bonne valeur agronomique, et cette introgression est suivie par des marqueurs moléculaires bordant la zone des QTL que l'on veut introgresser.The other possibility for assessing the effect of alleles at QTL is the crossing approach: - either by crossing between two lines or varieties; - either by using a series of related or diallel crosses. The principle is however the same for these two types of crosses since the effect of the allele at QTL is evaluated by comparison of the digestibility value of the descendants of the crosses. In a second step, two varieties (or two individuals from descendants), respectively having an allele favorable to a good digestibility value for the QTL of chromosome 1 and for the QTL of chromosome 5, are crossed in order to obtain an individual from the offspring of this new cross which contains these two alleles. Molecular markers belonging to these two chromosomal zones are then used to identify this. individual. This individual can then be fixed by successive self-fertilization, making sure not to lose the two alleles thanks to the molecular markers. From this individual or from parental lines of this individual, the two alleles can also be introgressed in a line or variety presenting a good agronomic value, and this introgression is followed by molecular markers bordering the zone of QTLs which it is desired to introgress.
B. Par transgénèse :B. By transgenesis:
On peut également obtenir des plantes plus digestibles par voie de transgénèse, en jouant sur le niveau d'expression des gènes codant pour des enzymes impliquées dans la biosynthèse de la lignine (surexpression de la F5H et sous-expression des autres par exemple) ou en introduisant une ou plusieurs séquences de gènes intervenant dans la biosynthèse de la lignine, séquences qui correspondront à des alleles associés à des valeurs de digestibilité fortes. Des cassettes d'expression sont construites à partir des séquences localisées dans les régions chromosomiques définies selon la présente invention, liées génétiquement à des séquences régulatrices de type promotrices (promoteurs forts ou spécifiques de tissus à forte lignification) et terminatrices (poly A nos par exemple). Ces cassettes sont ensuite intégrées dans des vecteurs d'expression contenant également des marqueurs de sélection pour la transformation, suivant les techniques standards décrites par exemple dans Sambrook et al. (1989). Ces vecteurs sont enfin utilisés pour transformer les cellules végétales selon les techniques connues de l'homme du métier. On peut citer notamment la transformation par canon à particules et la transformation par Agrobacterium, décrites ci-dessous.We can also obtain more digestible plants by transgenesis, by playing on the level of expression of genes coding for enzymes involved in the biosynthesis of lignin (overexpression of F5H and under-expression of others for example) or by introducing one or more sequences of genes involved in the biosynthesis of lignin, sequences which will correspond to alleles associated with strong digestibility values. Expression cassettes are constructed from the sequences localized in the chromosomal regions defined according to the present invention, genetically linked to regulatory sequences of promoter type (strong promoters or specific for highly lignification tissues) and terminators (poly A nos for example ). These cassettes are then integrated into expression vectors also containing selection markers for transformation, according to the standard techniques described for example in Sambrook et al. (1989). These vectors are finally used to transform plant cells according to techniques known to those skilled in the art. Mention may in particular be made of transformation by particle gun and transformation by Agrobacterium, described below.
Il est envisageable d'utiliser des promoteurs tissus ou organes spécifiques qui permettent l'expression des transgènes et donc l'amélioration de la digestibilité dans une partie seulement de la plantes. Sachant que l'augmentation de digestibilité liée à la diminution de la quantité de lignine est accompagnées dans certain cas par une augmentation de la sensibilité à la verse (mutant bm3 = mutant du gène CAOMT), il est avantageux d'augmenter la digestibilité par expression des transgènes dans toute la plante sauf dans la tige.
B-1 Canon à particulesIt is conceivable to use specific tissue or organ promoters which allow the expression of transgenes and therefore the improvement of digestibility in only part of the plant. Knowing that the increase in digestibility linked to the decrease in the amount of lignin is accompanied in certain cases by an increase in sensitivity to lodging (mutant bm3 = mutant of the CAOMT gene), it is advantageous to increase the digestibility by expression transgenes throughout the plant except the stem. B-1 Particle gun
La méthode utilisée repose sur l'utilisation d'un canon à particules identique à celui décrit par J. Finer (1992). Les cellules cibles sont des cellules indifférenciées en divisions rapides ayant conservé une aptitude à la régénération de plantes entières. Ce type de cellules compose le cal embryogène (dit de type II) de maïs. Ces cals sont obtenus à partir d'embryons immatures de génotype Hill selon la méthode et sur les milieux décrits par Armstrong (Maize Handbook ; 1994 M. Freeling, V. Walbot Eds ; pp.665-671 ). Ces fragments des cals d'une surface de 10 à 20 mm2 ont été disposés, 4h avant bombardement, à raison de 16 fragments par boite au centre d'une boîte de Pétri contenant un milieu de culture identique au milieu d'initiation, additionné de 0,2 M de maπnitol + 0,2 M de sorbitol. Des plasmides porteurs des gènes à introduire, en l'occurrence 4CL1 , 4CL2, CAD, CCR et ou F5H, sont purifiés sur colonne QiagenR en suivant les instructions du fabricant. Ils sont ensuite précipités sur des particules de tungstène (M10) en suivant le protocole décrit par Klein (1987). Les particules ainsi enrobées sont projetées vers les cellules cibles à l'aide du canon et selon le protocole décrits par J. Finer (1992). Les boîtes de cals ainsi bombardées sont ensuite scellées à l'aide de ScellofraisR puis cultivées à l'obscurité à 27°C. Le premier repiquage a lieu 24h après, puis tous les quinze jours pendant 3 mois sur milieu identique au milieu d'initiation additionné d'un agent sélectif. On obtient après 3 mois ou parfois plus tôt, des cals dont la croissance n'est pas inhibée par l'agent sélectif, habituellement et majoritairement composés de cellules résultant de la division d'une cellule ayant intégré dans son patrimoine génétique une ou plusieurs copies du gène de sélection. La fréquence d'obtention de tels cals est d'environ 0,8 cal par boîte bombardée.The method used is based on the use of a particle gun identical to that described by J. Finer (1992). Target cells are undifferentiated cells in rapid divisions which have retained the ability to regenerate whole plants. This type of cell makes up the embryogenic callus (called type II) of corn. These calluses are obtained from immature Hill genotype embryos according to the method and on the media described by Armstrong (Maize Handbook; 1994 M. Freeling, V. Walbot Eds; pp.665-671). These callus fragments with an area of 10 to 20 mm 2 were placed, 4 hours before bombardment, at the rate of 16 fragments per dish in the center of a Petri dish containing a culture medium identical to the initiation medium, added 0.2 M maπnitol + 0.2 M sorbitol. Plasmids carrying the genes to be introduced, in this case 4CL1, 4CL2, CAD, CCR and or F5H, are purified on a Qiagen R column according to the manufacturer's instructions. They are then precipitated on tungsten particles (M10) following the protocol described by Klein (1987). The particles thus coated are projected towards the target cells using the cannon and according to the protocol described by J. Finer (1992). The callus boxes thus bombarded are then sealed using Scellofrais R and then grown in the dark at 27 ° C. The first subculture takes place 24 hours later, then every two weeks for 3 months on medium identical to the initiation medium added with a selective agent. Calls are obtained after 3 months or sometimes earlier, calluses whose growth is not inhibited by the selective agent, usually and mainly composed of cells resulting from the division of a cell having integrated into its genetic heritage one or more copies of the selection gene. The frequency of obtaining such calluses is approximately 0.8 cal per bombarded box.
Ces cals sont identifiés, individualisés, amplifiés puis cultivés de façon à régénérer des plantules, en modifiant l'équilibre hormonal et osmotique des cellules selon la méthode décrite par Vain et al. (1989). Ces plantes sont ensuite acclimatées en serre où elles peuvent être croisées pour l'obtention d'hybrides ou autofécondées.
B-2. Transformation par AprobacteriumThese calluses are identified, individualized, amplified and then cultivated so as to regenerate seedlings, by modifying the hormonal and osmotic balance of the cells according to the method described by Vain et al. (1989). These plants are then acclimatized in the greenhouse where they can be crossed to obtain hybrids or self-fertilized. B-2. Transformation by Aprobacterium
Une autre technique de transformation utilisable dans le cadre de l'invention utilise Agrobacterium tumefaciens, selon le protocole décrit par Ishida et al (1996), notamment à partir d'embryons immatures de 1 0 jours après la fécondation. Tous les milieux utilisés sont référencés dans la référence citée. La transformation débute avec une phase de co-culture où les embryons immatures des plantes de maïs sont mis en contact pendant au moins 5 minutes avec Agrobacterium tumefaciens LBA 4404 contenant les vecteurs superbinaires. Le plasmide superbinaire est le résultat d'une recombinaison homologue entre un vecteur intermédiaire porteur de l'ADN-T contenant le gène d'intérêt (en l'occurrence 4CL1 , 4CL2, CAD, CCR et/ou F5H), et le vecteur pSB 1 de Japan Tobacco (EP 672 752) qui contient : les gènes virB et virG du plasmide pTiBo542 présent dans la souche supervirulente A281 d'Agrobacterium tumefaciens (ATCC 37349) et une région homologue retrouvée dans le vecteur intermédiaire permettant cette recombinaison homologue. Les embryons sont ensuite placés sur milieu LSAs pendant 3 jours à l'obscurité et à 25°C. Une première sélection est effectuée sur les cals transformés : les cals embryogènes sont transférés sur milieu LSD5 contenant de la phosphinotricine à 5 mg/l et de la céfotaxime à 250 mg/l (élimination ou limitation de la contamination par Agrobacterium tumefaciens). Cette étape est menée 2 semaines à l'obscurité et à 25°C. La deuxième étape de sélection est réalisée par transfert des embryons qui se sont développés sur milieu LSD5, sur milieu LSD10 (phosphinotricine à 10 mg/l) en présence de céfotaxime, pendant 3 semaines dans les mêmes conditions que précédemment. La troisième étape de sélection consiste à exciser les cals de type I (fragments de 1 à 2 mm) et à les transférer 3 semaines à l'obscurité et à 25°C sur milieu LSD 10 en présence de céfotaxime.Another transformation technique which can be used in the context of the invention uses Agrobacterium tumefaciens, according to the protocol described by Ishida et al (1996), in particular from immature embryos 10 days after fertilization. All the media used are referenced in the cited reference. The transformation begins with a co-culture phase where the immature embryos of the corn plants are brought into contact for at least 5 minutes with Agrobacterium tumefaciens LBA 4404 containing the superbinary vectors. The superbinary plasmid is the result of homologous recombination between an intermediate vector carrying the T-DNA containing the gene of interest (in this case 4CL1, 4CL2, CAD, CCR and / or F5H), and the vector pSB 1 from Japan Tobacco (EP 672 752) which contains: the virB and virG genes of the plasmid pTiBo542 present in the supervirulent strain A281 of Agrobacterium tumefaciens (ATCC 37349) and a homologous region found in the intermediate vector allowing this homologous recombination. The embryos are then placed on LSAs medium for 3 days in the dark and at 25 ° C. A first selection is made on the transformed calluses: the embryogenic calluses are transferred to LSD5 medium containing phosphinotricin at 5 mg / l and cefotaxime at 250 mg / l (elimination or limitation of contamination by Agrobacterium tumefaciens). This stage is carried out for 2 weeks in the dark and at 25 ° C. The second selection step is carried out by transfer of the embryos which have developed on LSD5 medium, on LSD10 medium (phosphinotricin at 10 mg / l) in the presence of cefotaxime, for 3 weeks under the same conditions as above. The third selection step consists in excising the type I calluses (fragments of 1 to 2 mm) and transferring them 3 weeks in the dark and at 25 ° C to LSD 10 medium in the presence of cefotaxime.
La régénération des plantules est effectuée en excisant les cals de type I qui ont proliféré et en les transférant sur milieu LSZ en présence de phosphinotricine à 5 mg/l et de céfotaxime pendant 2 semaines à 22°C et sous lumière continue.The regeneration of the seedlings is carried out by excising the type I calluses which have proliferated and by transferring them to LSZ medium in the presence of phosphinotricin at 5 mg / l and cefotaxime for 2 weeks at 22 ° C. and under continuous light.
Les plantules ayant régénéré sont transférées sur milieu RM + G2 contenant 1 00mg/l d'Augmentin pendant 2 semaines à 22°C et sous
illumination continue pour l'étape de développement. Les plantes obtenues sont alors transférées au phytotron en vue de leur acclimatation.The regenerated seedlings are transferred to RM + G2 medium containing 1,00 mg / l of Augmentin for 2 weeks at 22 ° C. and under continuous illumination for the development stage. The plants obtained are then transferred to the phytotron for their acclimatization.
Exemple 5 :Example 5:
Utilisation de la population issue de l'hybride Symphony®.Use of the population resulting from the Symphony® hybrid.
De façon analogue, l'invention peut être réalisée en utilisant comme matériel végétal de départ une population issue de l'hybride Symphony®.
Similarly, the invention can be carried out using as starting plant material a population resulting from the Symphony® hybrid.
REFERENCES BIBLIOGRAPHIQUESBIBLIOGRAPHICAL REFERENCES
- Allina SM et al, 4-Coumarate:coenzyme A ligase in hybrid poplar. Properties of native enzymes, cDNA cloning, and analysis of recombinant enzymes. Plant Physiol, 1998 Feb, 116(2):743-54- Allina SM et al, 4-Coumarate: coenzyme A ligase in hybrid poplar. Properties of native enzymes, cDNA cloning, and analysis of recombinant enzymes. Plant Physiol, 1998 Feb, 116 (2): 743-54
- Aufrere J et al, Predicting organic matter digestibility of forage by two pepsin-cellulase methods. XVI Congrès International des herbages, 1989.- Aufrere J et al, Predicting organic matter digestibility of drilling by two pepsin-cellulase methods. XVI International Grassland Congress, 1989.
- Beavis WD et al : 1991 Quantitative trait loci for plant height in four maize popoulations and their associations with qualitative genetic loci- Beavis WD et al: 1991 Quantitative trait loci for plant height in four maize popoulations and their associations with qualitative genetic loci
Then , Appl. Gent. 83:141-145.Then, Appl. Gent. 83: 141-145.
- Civardi L et al, Molecular cloning and characterisation of two cDNAs encoding enzymes required for secondary cell wall biosynthesis in Maize. In Cellular intégration of signalling pathways in plant development. Edited by Schiavo FL, Last RL, Morelli G and Raikhel NV - Springer-Verlag Berlin Heidelberg, 1998.D de Vienne, 1998, Les marqueurs moléculaires en génétique et biotechnologies végétales, Mieux comprendre, Editions INRA- Civardi L et al, Molecular cloning and characterization of two cDNAs encoding enzymes required for secondary cell wall biosynthesis in Maize. In Cellular integration of signaling pathways in plant development. Edited by Schiavo FL, Last RL, Morelli G and Raikhel NV - Springer-Verlag Berlin Heidelberg, 1998.D de Vienne, 1998, Molecular markers in plant genetics and biotechnology, Better understanding, INRA publications
- T. Helentjaris, 1987 A linkage map for maize based on RFLPs. Trends Genet 3 .217-221 - Hu WJ et al, Compartmentalized expression of two structurally and functionally distinct 4-coumarate:CoA ligase gènes in aspen (Populus tremuloides). Proc Natl Acad Sci U S A, 1998 Apr 28, 95(9):5407-12- T. Helentjaris, 1987 A linkage map for maize based on RFLPs. Trends Genet 3. 217-221 - Hu WJ et al, Compartmentalized expression of two structurally and functionally distinct 4-coumarate: CoA ligase genes in aspen (Populus tremuloides). Proc Natl Acad Sci U S A, 1998 Apr 28, 95 (9): 5407-12
- Jung HG et al, Characteristics of plant cell walls affecting intake and digestibility of forages by ruminants. J Anim Sci, 1995 Sep, 73(9):2774-90 - Lander et Botstein 1989 Mapping mendelian factors underiying quantitative traits using RFLP linkage maps. Genetics 121 :185-199- Jung HG et al, Characteristics of plant cell walls affecting intake and digestibility of forages by ruminants. J Anim Sci, 1995 Sep, 73 (9): 2774-90 - Lander and Botstein 1989 Mapping mendelian factors underiying quantitative traits using RFLP linkage maps. Genetics 121: 185-199
-Lefort-Buson M, De nouvelles perspectives pour l'analyse génétique des caractères quantitatifs. I- A la recherche des loci importants. Biofutur (1990) 91 : 30-37 - Lincoln ES et al, Constructing genetic linkage map with-Lefort-Buson M, New perspectives for the genetic analysis of quantitative traits. I- In search of important loci. Biofutur (1990) 91: 30-37 - Lincoln ES et al, Constructing genetic linkage map with
Mapmaker/EXP version 3.0: a tutorial and référence manual. Whitehead Institute Technical Report, Cambridge, 3rd Edition, January, 1993.
- Lincoln ES et al, Mapping gènes controlling quantitative traits using Mapmaker/QTL version 1.1 : a tutorial and référence manual. Whitehead Institute Technical Report, Cambridge, 2nd Edition, January, 1993.Mapmaker / EXP version 3.0: a tutorial and reference manual. Whitehead Institute Technical Report, Cambridge, 3 rd Edition, January, 1993. - Lincoln ES et al, Mapping genes controlling quantitative traits using Mapmaker / QTL version 1.1: a tutorial and reference manual. Whitehead Institute Technical Report, Cambridge, 2 nd Edition, January, 1993.
- Lubberstedt T et al, QTL mapping in testcross of European Flint Lines of Maize: Il comparison across populations for forage traits. Crop Sci,- Lubberstedt T et al, QTL mapping in testcross of European Flint Lines of Maize: Il comparison across populations for drilling traits. Crop Sci,
1998 38:1278-1289.1998 38: 1278-1289.
- Lubberstedt T et al, QTL mapping in testcross of European Flint Lines of Maize: Il comparison of différent testers for forage quality traits. Crop Sci, 1997 37:1913-1922 - Menke KH et al, The estimation of the digestibility and metabolizable energy content of ruminant feedingstuff from the gas production when they are incubated with rumen liquor in vitro. J. Agric Sci (Cambridge), 1979, 93:217-222.- Lubberstedt T et al, QTL mapping in testcross of European Flint Lines of Maize: Il comparison of different testers for drilling quality traits. Crop Sci, 1997 37: 1913-1922 - Menke KH et al, The estimation of the digestibility and metabolizable energy content of ruminant feedingstuff from the gas production when they are incubated with rumen liquor in vitro. J. Agric Sci (Cambridge), 1979, 93: 217-222.
- Ronsin T et al, Use of NIRS détermination quality in a silage Maize breeding program. The proceedings of the Third International Near- Ronsin T et al, Use of NIRS determination quality in a silage Maize breeding program. The proceedings of the Third International Near
Infrared Spectroscopy Conférence. June 25-29, 1990 in Brussels - Belgium.Infrared Spectroscopy Conference. June 25-29, 1990 in Brussels - Belgium.
- Song KM, Assessment of the degree of restriction fragment length polymorphism in Brassica. Theoretical and Applied Genetics (1988) 75 : 833-840 - Tanksley ét al, (1982), Heredity, n° 49, pp 1 1 -25- Song KM, Assessment of the degree of restriction fragment length polymorphism in Brassica. Theoretical and Applied Genetics (1988) 75: 833-840 - Tanksley et al, (1982), Heredity, n ° 49, pp 1 1 -25
- Van Soest, Use of the détergents in the analysis of fribrous feeds. J. Assoc. Offic. Agric. Chem. 1963 46:825-835.- Van Soest, Use of the detergents in the analysis of fribrous feeds. J. Assoc. Offic. Agric. Chem. 1963 46: 825-835.
-Young ND et Tanksley SD,Graphics-based whole génome sélection using RFLPs. Current communications in molecular biology - Development and application of molecular markers to problem in plant genetics. (1989) : 123-129
-Young ND and Tanksley SD, Graphics-based whole genome selection using RFLPs. Current communications in molecular biology - Development and application of molecular markers to problem in plant genetics. (1989): 123-129
Claims
1. Région chromosomique identifiée comme étant un locus QTL de digestibilité du maïs, caractérisée en ce qu'elle est délimitée par les marqueurs UMC 67 et UMC 128 sur le chromosome 1 du maïs.1. Chromosome region identified as a QTL locus of corn digestibility, characterized in that it is delimited by the markers UMC 67 and UMC 128 on chromosome 1 of corn.
2. Région chromosomique identifiée comme étant un locus QTL de digestibilité du maïs, caractérisée en ce qu'elle est comprise entre les marqueurs UMC 67 et UMC 128 sur le chromosome 1 du maïs et qu'elle comprend le gène 4CL2 codant pour l'enzyme 4-coumarate CoA ligase 2 et le gène CCR codant pour l'enzyme Cynnamoyl CoA réductase.2. Chromosomal region identified as a QTL locus of corn digestibility, characterized in that it is between the markers UMC 67 and UMC 128 on the chromosome 1 of corn and that it comprises the gene 4CL2 coding for the enzyme 4-coumarate CoA ligase 2 and the CCR gene coding for the enzyme Cynnamoyl CoA reductase.
3. Région chromosomique identifiée comme étant un locus QTL de digestibilité du maïs, caractérisée en ce qu'elle est délimitée par les marqueurs bnlg 1046 et bnlg 609 sur le chromosome 5 du maïs.3. Chromosome region identified as a QTL locus of corn digestibility, characterized in that it is delimited by the markers bnlg 1046 and bnlg 609 on chromosome 5 of corn.
4. Région chromosomique identifiée comme étant un locus QTL de digestibilité du maïs, caractérisée en ce qu'elle est comprise entre les marqueurs bnlg 1046 et bnlg 609 sur le chromosome 5 du maïs et qu'elle comprend le gène 4CL1 codant pour l'enzyme 4-coumarate CoA ligase 1 et le gène CAD codant pour l'enzyme cinnamyl alcool deshydrogénase et le gène F5H codant pour l'enzyme ferulate 5-hydroxylase.4. Chromosomal Region identified as a locus QTL corn digestibility, characterized in that it is between the markers bnlg 1046 and bnlg 609 on chromosome 5 of the corn and it comprises the 4CL1 gene encoding the enzyme 4-coumarate CoA ligase 1 and the CAD gene coding for the enzyme cinnamyl alcohol dehydrogenase and the gene F5H coding for the enzyme ferulate 5-hydroxylase.
5. Utilisation d'au moins une sonde ou amorce nucléique obtenue à partir de tout ou partie de la région chromosomique telle que définie dans l'une quelconque des revendications 1 à 4 pour l'identification chez un maïs du génotype au moins partiellement responsable d'une digestibilité élevée.5. Use of at least one nucleic probe or primer obtained from all or part of the chromosomal region as defined in any one of claims 1 to 4 for the identification in a corn of the genotype at least partially responsible for '' high digestibility.
6. Utilisation d'au moins une sonde ou amorce nucléique obtenue à partir de tout ou partie de la région chromosomique telle que définie dans l'une quelconque des revendications 1 à 4 pour le suivi de l'introgression du QTL de digestibilité du maïs, dans un programme de sélection de maïs assistée par marqueurs.6. Use of at least one nucleic probe or primer obtained from all or part of the chromosomal region as defined in any one of claims 1 to 4 for monitoring introgression corn digestibility QTL, in a marker-assisted corn selection program.
7. Méthode de détermination d'une corrélation entre ; l'haplotype des régions chromosomiques identifiées comme étant un locus QTL de digestibilité d'un maïs et sa valeur de digestibilité, comprenant : a) l'haplotypage de tout ou partie de la région chromosomique identifiée comme étant un locus QTL de digestibilité, telle que définie dans l'une quelconque des revendications 1 à 4, cette étape d'haplotypage étant 0 réalisée sur des individus appartenant à la descendance des maïs sur lesquels les régions chromosomiques de l'une des revendications 1 à 4 ont été identifiées comme étant un locus QTL de digestibilité du maïs ; et/ou b) l'haplotypage de tout ou partie de la région chromosomique du locus QTL de digestibilité telle que définie dans l'une 5 quelconque des revendications 1 à 4, cette étape d'haplotypage étant réalisée sur des individus qui ne sont pas apparentés auxdits individus de l'étape (a) ; c) la détermination de la valeur de digestibilité de l'ensemble desdits individus ; d) l'établissement d'une corrélation entre la valeur de 0 digestibilité et les profils haplotypiques obtenus.7. Method for determining a correlation between; the haplotype of the chromosomal regions identified as being a QTL locus of digestibility of a maize and its digestibility value, comprising: a) the haplotyping of all or part of the chromosomal region identified as being a QTL locus of digestibility, such as defined in any one of claims 1 to 4, this haplotyping step being 0 carried out on individuals belonging to the progeny of maize on which the chromosomal regions of one of claims 1 to 4 have been identified as being a locus Corn digestibility QTL; and / or b) haplotyping all or part of the chromosomal region of the QTL locus of digestibility as defined in any one of claims 1 to 4, this haplotyping step being carried out on individuals who are not related to said individuals of step (a); c) determining the digestibility value of all of said individuals; d) establishing a correlation between the value of 0 digestibility and the haplotypic profiles obtained.
8. Méthode de détermination prédictive de la valeur de digestibilité d'un maïs, comprenant : a) l'haplotypage de tout ou partie de la région chromosomique 5 identifiée comme étant un locus QTL de digestibilité, telle que définie dans l'une quelconque des revendications 1 à 4; b) la détermination prédictive de la valeur de digestibilité dudit maïs, au moyen de la corrélation établie à la revendication 7.8. A method for predictive determination of the digestibility value of a corn, comprising: a) haplotyping of all or part of the chromosomal region 5 identified as being a QTL locus of digestibility, as defined in any one of claims 1 to 4; b) the predictive determination of the digestibility value of said maize, by means of the correlation established in claim 7.
0 9. Méthode de sélection de maïs à digestibilité élevée, comprenant : **> ?0 9. High digestibility corn selection method, comprising: ** >?
l'haplotypage de tout ou partie de la région chromosomique identifiée comme étant un locus QTL de digestibilité, telle que définie dans l'une quelconque des revendications 1 à 4, la sélection parmi ces individus des plantes qui présentent une valeur prédictive de digestibilité élevée déterminée par la méthode de la revendication 8.haplotyping all or part of the chromosomal region identified as a QTL locus of digestibility, as defined in any one of claims 1 to 4, the selection among these individuals of plants which have a predicable predictive value for determined high digestibility by the method of claim 8.
10. Méthode de sélection de maïs ayant un degré de digestibilité élevé, comprenant : - le génotypage d'individus au moyen de sondes ou amorces nucléiques obtenues à partir de tout ou partie de la région chromosomique telle que définie dans l'une quelconque des revendications 1 à 4 ou bordant celle-ci, la sélection parmi ces individus des plantes qui comprennent une fréquence élevée d'allèles favorables associés à la digestibilité.10. A method of selecting maize having a high degree of digestibility, comprising: - the genotyping of individuals by means of nucleic probes or primers obtained from all or part of the chromosomal region as defined in any one of the claims 1 to 4 or bordering it, the selection among these individuals of plants which include a high frequency of favorable alleles associated with digestibility.
11. Méthode d'obtention de maïs génétiquement transformé ayant une digestibilité élevée, ladite méthode comprenant :11. Method for obtaining genetically transformed corn having a high digestibility, said method comprising:
(i) la transformation d'une cellule de plant de maïs avec au moins deux séquences nucleotidiques codant pour des enzymes différentes l'une de l'autre, lesdites séquences étant choisies parmi une séquence d'un allèle favorable à une digestibilité élevée codant pour la CCR, une séquence d'un allèle favorable à une digestibilité élevée codant pour la 4CL2, une séquence d'un allèle favorable à une digestibilité élevée pour la 4CL1 , une séquence d'un allèle favorable à une digestibilité élevée codant pour la CAD, et une séquence d'un allèle favorable à une digestibilité élevée codant pour la F5H, lesdites séquences étant portées par un ou plusieurs acides nucléiques ; et(i) the transformation of a corn plant cell with at least two nucleotide sequences coding for enzymes different from each other, said sequences being chosen from a sequence of an allele favorable to high digestibility coding for CCR, a sequence of an allele favorable to high digestibility coding for 4CL2, a sequence of an allele favorable to high digestibility for 4CL1, a sequence of an allele favorable to high digestibility coding for CAD, and a sequence of an allele favorable to high digestibility coding for F5H, said sequences being carried by one or more nucleic acids; and
(ii) la régénération d'une plante transgénique à partir de la cellule ainsi transformée. (ii) the regeneration of a transgenic plant from the cell thus transformed.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR0001152A FR2804970B1 (en) | 2000-01-28 | 2000-01-28 | IDENTIFICATION OF GENES ASSOCIATED WITH A QTL LOCUS OF MAIZE DIGESTIBILITY |
| FR0001152 | 2000-01-28 | ||
| PCT/FR2001/000272 WO2001055395A1 (en) | 2000-01-28 | 2001-01-29 | Identifying genes associated with a qtl corn digestibility locus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP1250440A1 true EP1250440A1 (en) | 2002-10-23 |
Family
ID=8846454
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP01903991A Withdrawn EP1250440A1 (en) | 2000-01-28 | 2001-01-29 | Identifying genes associated with a qtl corn digestibility locus |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US20030175732A1 (en) |
| EP (1) | EP1250440A1 (en) |
| AU (1) | AU2001231932A1 (en) |
| CA (1) | CA2398633A1 (en) |
| FR (1) | FR2804970B1 (en) |
| WO (1) | WO2001055395A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2465917C (en) | 2001-11-07 | 2012-09-25 | Genesis Research And Development Corporation Limited | Compositions from the grasses lolium perenne and festuca arundinacea |
| CN102458099B (en) | 2009-06-05 | 2015-08-19 | 佛罗里达大学研究基金公司 | The separation of sugarcane Lignin biosynthesis gene and targeted inhibition |
| PL3560330T3 (en) | 2018-04-24 | 2022-09-19 | KWS SAAT SE & Co. KGaA | Plants with improved digestibility and marker haplotypes |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU631562B2 (en) * | 1988-02-22 | 1992-12-03 | Pioneer Hi-Bred International, Inc. | Genetic linkages between agronomically important genes and restriction fragment length polymorphisms |
| GB9109063D0 (en) * | 1991-04-26 | 1991-06-12 | Ici Plc | Modification of lignin synthesis in plants |
| FR2739395B1 (en) * | 1995-10-03 | 1997-12-05 | Centre Nat Rech Scient | DNA SEQUENCE ENCODING A CINNAMOYL COA REDUCTASE, AND THEIR APPLICATIONS IN THE FIELD OF REGULATING LIGNIN CONTENT OF PLANTS |
| GB9521848D0 (en) * | 1995-10-25 | 1996-01-03 | Zeneca Ltd | Modification of lignin synthesis in plants |
| US5767080A (en) * | 1996-05-01 | 1998-06-16 | Cargill, Incorporated | Enhanced milk production in dairy cattle |
| CA2260907A1 (en) * | 1996-07-19 | 1998-01-29 | Purdue Research Foundation | Manipulation of lignin composition in plants using a tissue-specific promoter |
| TR200000547T2 (en) * | 1997-08-27 | 2001-05-21 | Pioneer Hi-Bred International, Inc. | Genes encoding enzymes for lignin biosynthesis and their use. |
| US6399855B1 (en) * | 1997-12-22 | 2002-06-04 | Pioneer Hi-Bred International, Inc. | QTL mapping in plant breeding populations |
-
2000
- 2000-01-28 FR FR0001152A patent/FR2804970B1/en not_active Expired - Fee Related
-
2001
- 2001-01-29 EP EP01903991A patent/EP1250440A1/en not_active Withdrawn
- 2001-01-29 WO PCT/FR2001/000272 patent/WO2001055395A1/en not_active Application Discontinuation
- 2001-01-29 AU AU2001231932A patent/AU2001231932A1/en not_active Abandoned
- 2001-01-29 US US10/182,113 patent/US20030175732A1/en not_active Abandoned
- 2001-01-29 CA CA002398633A patent/CA2398633A1/en not_active Abandoned
Non-Patent Citations (1)
| Title |
|---|
| See references of WO0155395A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2398633A1 (en) | 2001-08-02 |
| US20030175732A1 (en) | 2003-09-18 |
| AU2001231932A1 (en) | 2001-08-07 |
| WO2001055395A1 (en) | 2001-08-02 |
| FR2804970B1 (en) | 2004-06-25 |
| FR2804970A1 (en) | 2001-08-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Pan et al. | PpYUC11, a strong candidate gene for the stony hard phenotype in peach (Prunus persica L. Batsch), participates in IAA biosynthesis during fruit ripening | |
| Ogundiwin et al. | A fruit quality gene map of Prunus | |
| RU2745987C2 (en) | Methods and compositions for breeding brachytic corn plants | |
| CN113631722A (en) | Methods for identifying, selecting and producing southern corn rust resistant crops | |
| Gisbert et al. | A spontaneous eggplant (Solanum melongena L.) color mutant conditions anthocyanin-free fruit pigmentation | |
| Tang et al. | Breeding of a new variety of peanut with high-oleic-acid content and high-yield by marker-assisted backcrossing | |
| Sahu et al. | Molecular marker validation and identification of Fusarium wilt resistant chickpea genotypes | |
| EP2461666A1 (en) | Brassica plant for restoring fertility in an ogura cytoplasmic male-sterility system, method for producing same, and use of said plant | |
| US20210222189A1 (en) | Methods of identifying, selecting, and producing southern corn rust resistant crops | |
| EP3110832B1 (en) | Plants with increased yield and method for producing said plants | |
| WO2019002569A1 (en) | Method for breeding hybrid plants | |
| EP2087122B1 (en) | Maize with good digestibility and disease resistant | |
| WO2001055395A1 (en) | Identifying genes associated with a qtl corn digestibility locus | |
| Nashima et al. | Identification of Quantitative Trait Loci of Fruit Quality and Color in Pineapples | |
| FR2923985A1 (en) | MAES WITH INCREASED TOLERANCE IN FUNGAL DISEASES. | |
| Yaguchi et al. | Molecular and biochemical identification of alien chromosome additions in shallot (Allium cepa L. Aggregatum group) carrying extra chromosome (s) of bunching onion (A. fistulosum L.) | |
| US9309509B1 (en) | Methods and compositions for sweet corn sugary enhancer (SEI) gene | |
| US20220282338A1 (en) | Methods of identifying, selecting, and producing anthracnose stalk rot resistant crops | |
| US10093991B2 (en) | Compositions and methods for the detection of the shrunken2-R mutation in maize | |
| Julier et al. | Methodology of alfalfa breeding: a review of recent achievements. | |
| Kang et al. | Interaction of rice quantitative trait locus gw9. 1 with three grain shape genes | |
| Namkoong et al. | Application of genetic markers to forest tree species: Draft report to IPGRI of the project Developing Decision-making Strategies on priorities for conservation and use of forest genetic resources | |
| Asante et al. | Marker-assisted breeding for improving the cooking and eating quality of rice | |
| US20240065219A1 (en) | Novel loci in grapes | |
| RU2834673C2 (en) | Snp markers and selection for low fiber content in members of genus brassica |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
| 17P | Request for examination filed |
Effective date: 20020719 |
|
| AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR |
|
| AX | Request for extension of the european patent |
Free format text: AL;LT;LV;MK;RO;SI |
|
| RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: MURIGNEUX, ALAIN Inventor name: PUIGDOMENECH, PERE, CSIC CENTRO DE Inventor name: PEREZ, PASCUAL Inventor name: CIVARDI, LAURA Inventor name: MAES, TAMARA Inventor name: MARTINANT, JEAN-PIERRE Inventor name: TIXIER, MARIE-HELENE Inventor name: RIGAU, JOAN |
|
| STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
| 18D | Application deemed to be withdrawn |
Effective date: 20060529 |