WO2001055395A1 - Identification de genes associes a un locus qtl de digestibilite du maïs - Google Patents
Identification de genes associes a un locus qtl de digestibilite du maïs Download PDFInfo
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- WO2001055395A1 WO2001055395A1 PCT/FR2001/000272 FR0100272W WO0155395A1 WO 2001055395 A1 WO2001055395 A1 WO 2001055395A1 FR 0100272 W FR0100272 W FR 0100272W WO 0155395 A1 WO0155395 A1 WO 0155395A1
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- 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.
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AU2001231932A AU2001231932A1 (en) | 2000-01-28 | 2001-01-29 | Identifying genes associated with a qtl corn digestibility locus |
EP01903991A EP1250440A1 (fr) | 2000-01-28 | 2001-01-29 | Identification de genes associes a un locus qtl de digestibilite du ma s |
CA002398633A CA2398633A1 (fr) | 2000-01-28 | 2001-01-29 | Identification de genes associes a un locus qtl de digestibilite du mais |
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FR00/01152 | 2000-01-28 | ||
FR0001152A FR2804970B1 (fr) | 2000-01-28 | 2000-01-28 | Identification de genes associes a un locus qtl de digestibilite du mais |
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Cited By (2)
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US7154027B2 (en) | 2001-11-07 | 2006-12-26 | Jeroen Demmer | Compositions isolated from forage grasses and methods for their use |
WO2010141928A3 (fr) * | 2009-06-05 | 2011-04-28 | University Of Florida Research Foundation, Inc. | Isolement et suppression ciblée de gènes de la biosynthèse de lignine dans la canne à sucre |
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DK3560330T3 (da) * | 2018-04-24 | 2022-07-11 | Kws Saat Se & Co Kgaa | Planter med forbedret fordøjelighed og markørhaplotyper |
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WO1993005159A1 (fr) * | 1991-04-26 | 1993-03-18 | Zeneca Limited | Modification de la synthese de la lignine dans les plantes |
WO1997012982A1 (fr) * | 1995-10-03 | 1997-04-10 | Centre National De La Recherche Scientifique | SEQUENCES D'ADN CODANT POUR UNE CINNAMOYL CoA REDUCTASE, ET LEURS APPLICATIONS DANS LE DOMAINE DE LA REGULATION DES TENEURS EN LIGNINES DES PLANTES |
EP0804883A2 (fr) * | 1996-05-01 | 1997-11-05 | Cargill Incorporated | Produit alimentaire pour animaux |
WO1998003535A1 (fr) * | 1996-07-19 | 1998-01-29 | Purdue Research Foundation | Modification de la composition de la lignine dans des plantes au moyen d'un promoteur specifique d'un tissu |
US5866791A (en) * | 1995-10-25 | 1999-02-02 | Zeneca Limited | Modification of lignin synthesis in plants |
WO1999010498A2 (fr) * | 1997-08-27 | 1999-03-04 | Pioneer Hi-Bred International, Inc. | Genes codant des enzymes pour une biosynthese de lignine et leurs utilisations |
WO1999032661A1 (fr) * | 1997-12-22 | 1999-07-01 | Pioneer-Hi-Bred International, Inc. | Etablissement des cartographies des qtl dans des populations vegetales de selection |
-
2000
- 2000-01-28 FR FR0001152A patent/FR2804970B1/fr not_active Expired - Fee Related
-
2001
- 2001-01-29 US US10/182,113 patent/US20030175732A1/en not_active Abandoned
- 2001-01-29 WO PCT/FR2001/000272 patent/WO2001055395A1/fr not_active Application Discontinuation
- 2001-01-29 EP EP01903991A patent/EP1250440A1/fr not_active Withdrawn
- 2001-01-29 AU AU2001231932A patent/AU2001231932A1/en not_active Abandoned
- 2001-01-29 CA CA002398633A patent/CA2398633A1/fr not_active Abandoned
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WO1993005159A1 (fr) * | 1991-04-26 | 1993-03-18 | Zeneca Limited | Modification de la synthese de la lignine dans les plantes |
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US5866791A (en) * | 1995-10-25 | 1999-02-02 | Zeneca Limited | Modification of lignin synthesis in plants |
EP0804883A2 (fr) * | 1996-05-01 | 1997-11-05 | Cargill Incorporated | Produit alimentaire pour animaux |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7154027B2 (en) | 2001-11-07 | 2006-12-26 | Jeroen Demmer | Compositions isolated from forage grasses and methods for their use |
US7732661B2 (en) | 2001-11-07 | 2010-06-08 | Jeroen Demmer | Compositions isolated from forage grasses and methods for their use |
US8003849B2 (en) | 2001-11-07 | 2011-08-23 | Jeroen Demmer | Compositions isolated from forage grasses and methods for their use |
WO2010141928A3 (fr) * | 2009-06-05 | 2011-04-28 | University Of Florida Research Foundation, Inc. | Isolement et suppression ciblée de gènes de la biosynthèse de lignine dans la canne à sucre |
Also Published As
Publication number | Publication date |
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US20030175732A1 (en) | 2003-09-18 |
EP1250440A1 (fr) | 2002-10-23 |
CA2398633A1 (fr) | 2001-08-02 |
FR2804970B1 (fr) | 2004-06-25 |
FR2804970A1 (fr) | 2001-08-17 |
AU2001231932A1 (en) | 2001-08-07 |
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