FR2833615A1 - Evaluating digestibility of fodder plants, useful for strain selection, comprises detecting alleles of the cafeoyl coenzymeA 3-O-methyltransferase gene - Google Patents

Abstract

The invention concerns maize CCoAOMT2 gene polymorphism, and uses of said polymorphism to develop and enhance forage crop plant digestibility.

Description

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 The present invention relates to genetic markers associated with improved digestibility of forage plants, and in particular to variants of the second isoform of the 3-Methoyloyl-3-Methyl Transferase (CCoAOMT-2), which improves the digestibility of the plants which produce them. contain.

 An important factor limiting the digestibility of forage plants is related to the presence in the walls of plant cells, phenolic compounds, especially lignins. Lignins establish different types of linkages with other parietal constituents and form a tight mesh that impedes the accessibility of carbohydrates, main sources of energy for herbivores, to digestive enzymes.

 Therefore, one of the preferred ways of improving the quality of forage plants is the selection or genetic production of less lignified or modified lignin plants. Among the plants that have been the subject of the most studies for this purpose is silage maize, which is one of the most widely used fodder crops, particularly in the production sector. dairy.

 It has thus been observed that a decrease in the lignin content leads to a strong improvement in the digestibility of the silage corn, which results in an increase in the milk production and in the weight gain compared to the fed animals. with a less digestible variety (CLIL, Annales de zootechnies, 1995).

 Corn mutants known as brown midrib are known which have a greatly reduced lignin content (up to 40%); and a lignin of different structure compared to normal corn,. These maize, and especially those of the so-called bm3 lines whose gene coding for 0-methyltransferase caffeic acid is mutated (VIGNOLS et al., The Plant Cell, 7, 407-416, 1995), have a greatly increased digestibility compared with the maize wild type, and significantly improve milk production when used as fodder.

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 However, maize bm3 have disadvantages limiting their exploitation, such as a lower field yield, increased susceptibility to lodging, a lack of growth and vigor at the beginning of vegetation and a delay in flowering (BARRIERE and ARGILLIER, Agronomy, 13, 855-876, 1993).

 The obtaining of fodder plants and particularly maize, more digestible following a modification of the biosynthesis pathway of lignins, having good yields and little sensitive to various stresses (pathological, hydric, mechanical ...) is one of the preferred axes plant improvement.

 Lignins are insoluble polymers of 3 alcohol monomers or monolignols, derived from the phenylpropanoid route (NEISH, Constitution and Biosynthesis of Lignin, New York editions: Springer Verlag pp. 1-43,1968): alcohol coumaryl (H subunits), coniferyl alcohol (G subunits) and sinapyl alcohol (S subunits).

 O-methyl transferases (S-adenosyl-L-methionine O-methyltransferases, EC 2. 1. 1.6) play an important role in the biosynthesis of monolignols. Although the mechanisms involved in vivo in this biosynthetic pathway are not fully understood, it is generally considered that the formation of S and G subunits involves successive reactions of hydroxylation and O-methylation followed by the conversion of the carboxyl side chain to an alcohol function.

-différentes O-méthyltransférases : l'acide caféique O-méthyltransférase (COMT) ; la 5-hydroxyconiferyl aldéhyde 0-methyl transférase (AldOMT) ; les Caféoyl
Coenzyme A 3-0 méthyltransférases (CCoAOMT) - des hydroxycinnamate coenzyme A ligases (4CL) - une férulate 5-hydroxylase (F5H) à cytochrome P450 - et plusieurs isoformes de Cinnamoyl Co A réductase (CCR) et de Cinnamyl alcool déhydrogenase (CAD). The enzymes involved in this biosynthetic pathway include:

-different O-methyltransferases: caffeic acid O-methyltransferase (COMT); 5-hydroxyconiferyl aldehyde O-methyl transferase (AldOMT); the Caféoyl
Coenzyme A 3-0 methyltransferases (CCoAOMT) - hydroxycinnamate coenzyme A ligases (4CL) - a ferulate 5-hydroxylase (F5H) to cytochrome P450 - and several isoforms of Cinnamoyl Co A reductase (CCR) and Cinnamyl alcohol dehydrogenase (CAD) .

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 The properties of these various enzymes have been reviewed (BOUDET et al., New Phytol., 129, 203-236, 1995; DIXON et al., Phytochemistry, in press, 2001; WHETTEN et al., Annu. Plant Physiol Plant, Mol Biol., 49, 855-609, 1998, LI et al., J. Biol Chem., 275, 6537-6545, 2000).

 The Coenzyme-A O-methyltransferases play a major role in the biosynthesis pathway of monolignols and more particularly G and S subunits; they appear to occur during several steps of the lignin biosynthesis pathway (GUO et al., Plant Cell, 13, 73-88, 2001), and it has been observed that CCoAOMT activity is closely related to the lignification of the lignins. tissues in a number of dicotyledonous species (ZHONG et al., Plant Cell, 10,2033-2045, 1998).

 In maize, two different genes encoding two isoforms of CCoAOMT have been identified (CIVARDI et al., Plant Physiol., 120, 1206, 1999): the CCoAOMT gene (AJ242980) and the CCoAOMT2 gene (AJ242981).

 The sequence of the wild type CCoAOMT2 gene used as a reference in the disclosure of the present invention corresponds to accession number AJ242981 on the basis of GenBank, and to that of CIVARDI et al., Plant Physiol., 120, 1206, ( 1999). The CCoAOMT2 gene described by CIVARDI et al. was obtained from the variety W64A; its sequence is shown in Figure 1. The fragment shown comprises 1181 base pairs and has 4 exons and 3 introns. The 5 'non-coding region of this sequence is defined as 70 base pairs. The intron splice sites are: for intron 1 (79 bp) AGGT .... AGTA, for intron 2 (108 bp) TGGT .... AGGA, and for intron 3 ( 84 bp) CGGT .... AGAT. The sequence of this fragment is also represented in the attached sequence listing under the number SEQ ID NO: 1, and the sequence of its translation product is represented under the number SEQ ID NO: 2.

 The inventors have demonstrated the colocalization of the gene CCoAOMT2, located on chromosome 9, with a QTL of digestibility and a QTL of lignin content.

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 in the walls. They further identified mutations in the CCoAOMT2 gene creating polymorphic alleles associated with corn digestibility.

 Polymorphisms associated with increased digestibility of maize were identified on an allele of the CCoAOMT2 gene isolated from a maize line called F4.

This line F4 has a very high level of digestibility, close to that of the bm3 lines, and unlike these, produces lignins which are characterized by a S / G ratio significantly higher than that observed in the lignins from the varieties. classic corn silage.

 The allele of the CCoAOMT2 gene isolated from the F4 line is represented in the attached sequence listing under the number SEQ ID NO: 3.

 Some of the polymorphisms identified on this allele are located at the level of the coding regions of the gene, and result in mutations in the 3-methyloyl-3-methylated Coenzyme A (CCoAOMT-2) resulting from the expression of this gene. Others are located in the 5 'non-coding region and in the introns, particularly intron 3.

 The identification by the inventors of the involvement of CCoAOMT-2 in digestibility, as well as the identification in the CCoAOMT2 gene of polymorphisms associated with digestibility, provides a set of means for obtaining forage plants, in particular monocotyledons, and in particular maize with improved digestibility.

 The subject of the present invention is a method for evaluating the digestibility of a forage plant, and in particular maize, characterized in that it comprises the detection of the presence or absence, in biological material coming from said plant, an allele favorable to the digestibility of the gene CCoAOMT2.

 According to a preferred embodiment of the present invention, said digestibility-promoting CCoAOMT2 allele encodes a mutant Caféoyl Coenzyme A 3-0 methyltransferase-2 (CCoAOMT-2) comprising at least one of the following mutations:

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 a) the deletion of the Glu-Ala-Thr peptide sequence, situated between positions 6 and 10 of the sequence SEQ ID NO: 2; b) inserting the Asn-GlyAsn-Gly-Asn-Gly peptide sequence between positions 26 and 27 of the sequence SEQ ID NO: 2; c) substituting the His residue at position 37 of the sequence SEQ ID NO: 2 by an Arg residue; d) the substitution of the residue Asn at position 188 of the sequence SEQ ID NO: 2 by a residue Ser.

 Advantageously, said allele favorable to digestibility comprises at least mutation a) above.

 For the implementation of the present invention, the detection of the presence of a digestibility-friendly CCoAOMT2 gene allele can be carried out by analysis of the CCoAOMT-2 protein, to highlight mutations, such as mutations. a) to d) mentioned above, which result in the modification of the peptide sequence of this protein.

 It can also be carried out by analysis of polymorphisms of the CCoAOMT2 gene of the test plant, to determine if one or more of these polymorphisms are present in their form associated with the allele favorable to the desired digestibility.

 The polymorphisms mentioned above may be polymorphisms located in the coding regions of the CCoAOMT2 gene, and which result in modifications of the peptide sequence of the CCoAOMT-2 protein, and in particular by the mutations a) to d) mentioned above. above. They may also be polymorphisms located in the 5 'non-coding region or in the introns of the CCoAOMT2 gene, or polymorphisms located in the coding regions but not resulting in a sequence modification of the CCoAOMT-2 protein. Indeed, although these polymorphisms do not appear to be directly involved in the CCoAOMT-2 mutation favorable for digestibility, they are nevertheless closely related to it, and their presence therefore constitutes an indicator of plant digestibility.

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In the context of the implementation of the present invention, it is thus possible to use one or more of the following polymorphisms, associated with the CCoAOMT2 gene allele of but favorable for the digestibility represented by the sequence SEQ ID NO: 3: insertion of 5 base pairs in the 5 'non-coding region between nucleotides 55 and 56 of the reference sequence SEQ ID NO: 1. The inserted fragment has the sequence ACTGC, and corresponds to positions 56 to 60 of the sequence SEQ ID NO: 3. The 5'non-coding region of the sequence allele
SEQ ID NO: 3 extends from nucleotide 1 to nucleotide 75 of this sequence, which corresponds to nucleotides
1 to 70 of the reference sequence SEQ ID NO: 1.

 Exon 1 of the sequence SEQ ID NO: 3 allele extends from nucleotide 76 to nucleotide 228 of this sequence, which corresponds to nucleotides 71 to 214 of the reference sequence SEQ ID NO: 1.

In exon 1, the sequence SEQ ID NO: 3 allele comprises the following polymorphisms: a deletion of 9 base pairs, corresponding to the sequence fragment located between the nucleotides 81 and
91 of the reference sequence SEQ ID NO: 1. The deleted fragment has the sequence: GGCGACCGA, and codes for the Glu-Ala-Thr peptide sequence absent in the sequence
SEQ ID NO: 4; an insertion of 18 base pairs between the nucleotides 129 and 130 of the reference sequence
SEQ ID NO: 1. The inserted fragment corresponds to positions 125 to 144 of the sequence SEQ ID NO: 3. This fragment has the sequence CAACGGCAACGGCAACGG, and codes for the Asn-Gly-Asn-Gly-Asn-Gly peptide sequence inserted into the sequence SEQ ID NO: 4; a CG substitution at position 178 of the reference sequence SEQ ID NO: 1, corresponding to position 192 of the sequence SEQ ID NO: 3; a substitution A-> G at position 180 of the reference ode sequence SEQ ID NO: 1, corresponding to the position

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194 of the sequence SEQ ID NO: 3. This substitution results in the substitution of the His residue at position 37 of the sequence SEQ ID NO: 2 by an Arg residue.

 Intron 1 of the sequence SEQ ID NO: 3 allele extends from nucleotide 229 to nucleotide 307 of this sequence, which corresponds to nucleotides 215 to 293 of the reference sequence SEQ ID NO: 1.

In intron 1, the sequence SEQ ID NO: 3 allele comprises the following polymorphisms: a GA substitution at position 227 of the reference sequence SEQ ID NO: 1, corresponding to position 241 of sequence SEQ ID NO : 3; a substitution A-> G at position 237 of the reference sequence SEQ ID NO: 1, corresponding to the position 251 of the sequence SEQ ID NO: 3; a CG- + TT substitution at positions 259-260 of the reference sequence SEQ ID NO: 1, corresponding to positions 273-274 of the sequence SEQ ID NO: 3;
Exon 2 of the sequence SEQ ID NO: 3 allele extends from nucleotide 308 to nucleotide 387 of this sequence, which corresponds to nucleotides 294 to 373 of the reference sequence SEQ ID NO: 1.

 In exon 2, the sequence SEQ ID NO: 3 allele has no polymorphism with respect to the reference sequence SEQ ID NO: 1.

 Intron 2 of the sequence SEQ ID NO: 3 allele extends from nucleotide 388 to nucleotide 495 of this sequence, which corresponds to nucleotides 374 to 481 of the reference sequence SEQ ID NO: 1.

 In intron 2, the sequence SEQ ID NO: 3 allele comprises the following polymorphism: an AG substitution at position 466 of the reference sequence SEQ ID NO: 1, corresponding to position 480 of the sequence SEQ ID NO : 3.

 Exon 3 of the sequence SEQ ID NO: 3 allele extends from nucleotide 496 to nucleotide 640, which corresponds to nucleotides 482 to 626 of the reference sequence SEQ ID NO: 1.

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 In exon 3, the sequence SEQ ID NO: 3 allele has no polymorphism with respect to the reference sequence SEQ ID NO: 1.

 Intron 3 of the sequence SEQ ID NO: 3 allele extends from nucleotide 641 to nucleotide 754, which corresponds to nucleotides 627 to 710 of the reference sequence SEQ ID NO: 1.

In intron 3, the sequence SEQ ID NO: 3 allele comprises the following polymorphisms: a 15 base pair insertion between the nucleotides 631 and 632 of the SEQ reference sequence
ID NO: 1. The fragment inserted corresponds to the positions
646 to 660 of the sequence SEQ ID NO: 3. This fragment has for sequence: TGCCCCTTTTCTCTCT.

an insertion of 18 base pairs between nucleotides 703 and 704 of the SEQ reference sequence
ID NO: 1. The fragment inserted corresponds to the positions
730 to 747 of the sequence SEQ ID NO: 3. This fragment has the following sequence: CTCTCTGTTGCTCGTCCC.

a deletion of 3 base pairs, corresponding to the sequence fragment located between nucleotides 668 and
672 of the reference sequence SEQ ID NO: 1. The deleted fragment has for sequence: TGA; at least one CT substitution at position 635 or 648 of the reference sequence SEQ ID NO: 1, respectively corresponding to positions 664 and 677 of the sequence
SEQ ID NO: 3.

at least one substitution T--> C at position 638,672 or
692 of the reference sequence SEQ ID NO: 1, corresponding respectively to positions 667, 698 and
718 of the sequence SEQ ID NO: 3; an A o C substitution at position 667 of the reference sequence SEQ ID No. 1, corresponding to the position 696 of the sequence SEQ ID NO: 3; a substitution C-G at position 698 of the reference sequence SEQ ID No. 1, corresponding to position 724 of the sequence SEQ ID NO: 3;

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- a TG - ") 0 CC substitution at the positions 664-665 of the reference sequence SEQ ID Nol, corresponding to the positions 693-694 of the sequence SEQ ID NO: 3;
Exon 4 of the sequence SEQ ID NO: 3 allele extends from nucleotide 755 to nucleotide 1180, which corresponds to nucleotides 711 to 1136 of the reference sequence SEQ ID NO: 1.

 In exon 4, the sequence SEQ ID NO: 3 allele comprises the following polymorphism: a substitution A-> G at position 904 of the reference sequence SEQ ID NO: 1, corresponding to position 948 of the sequence SEQ ID NO: 3. This substitution results in the substitution of the residue Asn at position 188 of the sequence SEQ ID NO: 2 by a residue Ser.

 Polymorphisms whose use in the context of the implementation of the process according to the invention is particularly advantageous are the following: a deletion of 9 base pairs between nucleotides 81 and 91 of the reference sequence SEQ ID NO 1. an 18 base pair insertion, sequence CAACGGCAACGGCAACGG, between nucleotides 129 and 130 of the reference sequence SEQ ID NO: 1.

 For example, it is possible to detect in a plant the presence of a CCoAOMT-2 polymorphism in its form associated with a digestibility-friendly allele, from a sample containing genetic material from the test plant, by hybridization. selective polynucleotide probe specific for this allelic form.

 As the allele-specific probe of a polymorphism, any polynucleotide probe which, under appropriate stringency conditions, is capable of hybridizing selectively to any allele of the CCoAOMT-2 gene containing this allelic form, is defined here as an allelic-specific probe of a polymorphism, compared to other alleles of the same gene, and related sequence genes. This selective hybridization results in a significantly greater hybridization signal with said allele than with the other alleles of the same gene or the

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 related sequence genes, and preferably by the presence of a hybridization signal with said allele and the absence of hybridization signal with other alleles of the same gene or related sequence genes.

 The general principles of design and use of specific probes allowing the analysis of polymorphisms concerning only a small number of nucleotides, or even a single nucleotide, were initially described by SAIKI et al. (Nature, 324, 163-166, 1986), and are well known in themselves.

 Stringency conditions suitable for selective hybridization can be readily determined by those skilled in the art for a given polynucleotide; they depend in particular on the length of the polynucleotide, its composition in bases, and the percentage of differences between the alleles to be discriminated, in the portion of the gene targeted by the hybridization.

 For example, the CCoAOMT2 gene or a region thereof comprising at least one of the polymorphisms defined above can be amplified from genetic material derived from the test plant, and the presence or absence, in the amplification product, the form of said polymorphism associated with the desired allele. In this case, the primers and the amplification conditions used will be defined so as to allow the amplification of the different CCoAOMT2 alleles.

 The detection of the form of the polymorphism associated with the desired allele can for example be carried out using a polynucleotide probe according to the invention, as indicated above. It can also be carried out, especially when the polymorphism is an insertion or a deletion of several nucleotides, on the basis of the size of the amplification fragments.

 Alternatively, selective amplification of the desired allele can be carried out using at least one amplification primer specific for this allele. The amplification conditions will be defined according to the chosen primer, so that said primer does not hybridize

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 with its complementary sequence present in the desired allele. Under these conditions, amplification is only observed if this allele is present in the genetic material analyzed. The second primer may be common to different alleles, or specific to the desired allele.

 The subject of the present invention is also a mutant CCoAOMT-2, characterized in that it differs from CCoAOMT-2 of sequence SEQ ID NO: 2 by at least one of the following mutations: a) the deletion of the peptide sequence Glu-Ala-Thr between positions 6 and 10 of the sequence SEQ ID NO: 2; b) inserting the Asn-GlyAsn-Gly-Asn-Gly peptide sequence between positions 26 and 27 of the sequence SEQ ID NO: 2; c) substituting the His residue at position 37 of the sequence SEQ ID NO: 2 by an Arg residue; d) the substitution of the residue Asn at position 188 of the sequence SEQ ID NO: 2 by a residue Ser.

 Preferably, said mutant CCoAOMT-2 comprises at least mutation a).

 Most preferably, said mutant CCoAOMT-2 corresponds to the sequence SEQ ID NO: 4.

 The subject of the present invention is also an isolated polynucleotide encoding a mutant maize Coyloye A 3-0 methyltransferase-2 (CCoAOMT-2) according to the invention.

 According to a preferred embodiment of the present invention, said polynucleotide responds to a sequence chosen from: the genomic DNA sequence represented in the list of sequences in the appendix under the number SEQ ID NO: 3; this sequence is also shown in Figure 2.

 the cDNA sequence derived from said genomic sequence.

 The subject of the present invention is also any fragment of more than 10 bp, preferably of more than 15 bp, and quite preferably of more than 20 bp, of a

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 polynucleotide of sequence SEQ ID NO: 3, said fragment comprising at least one of the polymorphisms mentioned above, as well as the complement of said fragment.

 The polynucleotide of sequence SEQ ID NO: 3, or its fragments defined above, may in particular be used for detection in forage plants, in particular in monocotyledons, and in particular in maize, of a digestibility-friendly allele. of the gene coding for CCoAOMT-2. In particular, they can be used to obtain polynucleotide probes or amplification primers for this detection.

 For example, polynucleotide probes specific for the allelic form of a CCoAOMT-2 polymorphism present on the sequence SEQ ID NO: 3 allele may comprise in particular: the polynucleotide of sequence SEQ ID NO: 3 or its complement; fragments of said polynucleotide of more than 10 bp, preferably of more than 15 bp, and quite preferably of more than 20 bp, comprising at least one of the polymorphisms mentioned above, as well as the complementary ones of said fragments.

 As another example of using fragments of the polynucleotide of sequence SEQ ID NO: 3, in a method for detecting a polymorphism constituted by the deletion of several nucleotides, it is possible to amplify the region of exon 1 comprising a deletion of 9 bp in the SEQ ID NO: 3 allele, using the primers defined respectively by the sequences SEQ ID NO: 5 and SEQ ID NO: 6. The amplification product obtained from the genetic material obtained from a plant of the reference line W64A (comprising the SEQ ID NO: 1 allele), has a length of 85 base pairs, whereas the amplification product obtained from the genetic material derived from a plant of the F4 line (comprising the SEQ ID NO: 3 allele) has a length of 76 bp.

 The position of the primers SEQ ID NO: 5 and 6 is shown in Figure 2.

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 To perform a selective amplification of an allele of the CCoAOMT-2 gene containing the allelic form of a CCoAOMT-2 polymorphism present on the allele of sequence SEQ ID NO: 3, it is possible to use a pair of amplification primers. wherein at least one of the primers represents a fragment of at least 10 bp, preferably at least 15 bp, and most preferably at least 20 bp, of the polynucleotide of sequence SEQ ID NO: 3, said fragment comprising at least least one of the polymorphisms mentioned above, or the complementary of said fragment.

 The amplification conditions will be defined according to the chosen primer, as indicated above.

 For example, it is possible to use a pair of primers defined respectively by the sequences SEQ ID NO: 7 and SEQ ID NO: 8.

 The primer SEQ ID NO: 7 consists of a fragment of the sequence SEQ ID NO: 3 surrounding the location of the deletion of 9 bp in the first exon, to which were added 4 additional bases (the first 4 bases of the sequence SEQ ID NO: 7), to increase the Tm (melting temperature) of the primer, and thus the hybridization specificity.

 The primer SEQ ID NO: 8 is common to the different alleles of the CCoAOMT2 gene; it is located just before the stop codon of this gene.

 The position of the primers SEQ ID NO: 7 and 8 is shown in Figure 2.

 This pair of primers amplifies the sequence allele SEQ ID NO: 3, without amplifying the reference allele defined by the sequence SEQ ID NO: 1.

 The subject of the present invention is also oligonucleotide primers that can be used for carrying out the detection methods defined above.

 In particular, the subject of the present invention is: any pair of primers allowing the amplification of a region of the CCoAOMT2 gene comprising at least one of the polymorphisms mentioned above; as

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 for example, there will be mentioned the pair of primers defined by the sequences SEQ ID NO: 5 and SEQ ID NO: 6.

 any pair of primers of which at least one primer represents a fragment of at least 10 bp, preferably at least 15 bp, and most preferably at least 20 bp, of the polynucleotide of sequence SEQ ID NO: 3, comprising at least one of the polymorphisms mentioned above, or the complement of said fragment; by way of example, mention may be made of the pair of primers defined by the sequences SEQ ID NO: 7 and SEQ ID NO: 8.

 The subject of the invention is also a method for identifying a forage plant, and in particular a maize, comprising a CCoAOMT2 allele favorable to digestibility, characterized in that it comprises detecting the presence, in a sample derived from said plant of a mutant CCoAOMT-2 as defined above.

 The detection of a mutant CCoAOMT-2 according to the invention may be carried out, for example, using polyclonal or monoclonal antibodies selectively recognizing said protein, that is to say having no cross-reactions with d other maize proteins, and especially not cross-reactive with wild-type CCoAOMT-2 or with CCoAOMT-1.

 These antibodies, which are also part of the subject of the invention, can be easily obtained by conventional methods for preparing polyclonal or monoclonal antibodies, by immunizing an animal with a mutant CCoAOMT-2 according to the invention, or a fragment thereof carrying at least one of mutations a) to d) identified above, and in particular a fragment bearing at least mutation b), and selecting, from among the antibodies obtained, those which, under the same conditions reaction, form an antibody / antigen complex detectable with a mutant CCoAOMT-2 according to the invention, but not with other corn proteins, and in particular wild-type CCoAOMT-2 or CCoAOMT-1.

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 The antibodies according to the invention may optionally be labeled in order to allow the antibody / antigen complex to be revealed.

 The present invention also relates to kits for carrying out a method according to the invention, for detecting a CCoAOMT2 allele favorable to digestibility. These kits comprise, in addition to one or more reagents in accordance with the invention (in particular antibodies, polynucleotide probe or primers), appropriate means for carrying out the reaction used for the detection, and / or means suitable for the disclosure of the product. of this reaction.

For example, we will mention:
Kits comprising at least one polynucleotide probe according to the invention, optionally combined with reagents for carrying out a hybridization reaction, with means for detecting the hybrid formed, and / or with positive controls and / or negative hybridization controls;
Kits comprising at least one pair of primers according to the invention, optionally combined with reagents for carrying out an amplification reaction, with means for detecting the amplification product, and / or with positive controls and / or negative amplification controls;
Kits comprising at least one antibody according to the invention, optionally combined with reagents for carrying out an antibody / antigen reaction, with means for detecting an antibody / antigen complex, and / or with controls positive and / or negative reaction controls.

 These kits may comprise, in addition to the reagents according to the invention for detecting a digestibility-friendly CCoAOMT2 allele, other reagents for detecting other agronomic traits of interest.

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 The present invention can be implemented in the context of the selection of plants, in particular maize, having improved digestibility.

 It is of particular interest in the context of marker-assisted selection (SAM). This method of selection is much more effective than a simple phenotypic evaluation since all loci corresponding to different characters of interest can be analyzed together on a single DNA sample, regardless of the growing conditions of the plant from which it originated. the sample. Marker-assisted selection makes it possible to implement accelerated backcrossing techniques by using the existing link between a molecular marker and a gene of agronomic interest, in this case coding for CCoAOMT-2, to transfer it into different genotypes to provide them with increased digestibility.

 The present invention may also be implemented to monitor the integration of a CCoAOMT2 gene allele favorable to digestibility, for example in the context of introgression techniques by successive crosses between plants having this allele.

 The present invention can also be used in the context of phylogenetic studies, the characterization of genetic relationships among plant varieties, the identification of somatic crosses or hybridizations, the location of chromosomal segments affecting monogenic traits, cloning based on genetic maps, and identification of QTL and / or PQL.

 The polynucleotides according to the invention, and in particular the polynucleotide of sequence SEQ ID NO: 3, or their fragments comprising at least one of the polymorphisms defined above can also be used for the production, in recombinant form, of CCoAOMT- 2 mutant according to the invention, or fragments thereof, as well as to improve the digestibility of forage plants, particularly monocotyledons, and in particular maize, by

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 the expression in said plants of a mutant CCoAOMT-2 according to the invention.

 The subject of the present invention is thus: a recombinant expression cassette comprising a polynucleotide according to the invention or a fragment thereof, comprising at least one of the polymorphisms defined above placed under control of heterologous regulatory sequences transcription and translation (including transcription promoter and terminator). a recombinant vector resulting from the insertion, in a suitable host vector, of a polynucleotide according to the invention or of a fragment thereof, comprising at least one of the polymorphisms defined above, or of an expression cassette according to the invention.

 The present invention also encompasses a method for producing a recombinant polypeptide, characterized in that it comprises transforming a host cell, prokaryotic or eukaryotic, with a polynucleotide sequence coding for a mutant CCoAOMT according to the invention, or for a fragment thereof comprising at least one of mutations a) to d) identified above, and recovering said mutant CCoAOMT or said fragment produced by said cell.

 The present invention also encompasses host cells genetically transformed with a polynucleotide according to the invention.

 The host cells may be eukaryotic or prokaryotic cells. By way of examples, mention may be made of bacteria, in particular E. coli or Agrobacterium, yeasts, for example Saccharomyces, animal cells or, preferentially, plant cells.

 The present invention also encompasses transgenic plants genetically transformed with a polynucleotide according to the invention. It is preferentially monocotyledons, and in particular corn;

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Conventional techniques for constructing recombinant vectors, for transforming cells or host organisms, and for producing recombinant proteins, can be used for practicing the present invention.

 The selection of the host vector, and expression control sequences will be performed depending on the host cell or host organism chosen and the intended application.

 For example, for the production of recombinant polypeptides, host cells of bacteria, especially E. coli or yeasts, in particular Saccharomyces cerevisiae, are frequently used: many expression systems in one or other of these two organisms are known. in themselves and available commercially.

 For expression in plant cells or plants it is possible to use the endogenous promoter of the CCoAOMT2 gene.

des promoteurs inductibles, par exemple le promoteur Hsp70 qui est inductible par un choc thermique ; - des promoteurs tissu-spécifiques. It is also possible to use a heterologous promoter; very many promoters usable for the transformation of plant cells are known in themselves. By way of examples, mention may be made of: constitutive promoters, such as the CaMV 35S promoter, or its derivatives, or the ubiquitin promoter;

inducible promoters, for example the Hsp70 promoter which is inducible by heat shock; tissue-specific promoters.

 Among the transcription terminators that may be used, mention may in particular be made of the polyA 35S terminator of cauliflower mosaic virus, or the polyA NOS terminator, which corresponds to the 3 'non-coding region of the nopaline synthase gene of the Ti plasmid of Agrobacterium tumefaciens nopaline strain.

 The transformation of plant cells can be carried out by various methods such as, for example, the transfer of the polynucleotide of interest into the plant protoplasts after incubation of the latter in a solution.

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 polyethylene glycol in the presence of divalent cations (Ca 2+), electroporation (FROMM et al., Proc Nat Acad Sci., 82,5824, 1985), the use of a particle gun, particularly according to the method described by FINER et al. (Plant Cell Report, 11, 323-328, 1992), or cytoplasmic or nuclear microinjection (NEUHAUS et al., Theor Appl.

Genet., 75 (1), 30-36, 1987).

 The plant cells can also be infected by a bacterial cell host comprising the vector containing the sequence of interest. The host cell may be Agrobacterium tumefaciens (AN et al., Plant Physiol., 81, 86-91, 1986), or A. rhizogenes (GUERCHE et al., Mol. Gen.

Genet., 206,382, 1987). In this case, preferably, the transformation of the plant cells is carried out by the transfer of the T region of the extrachromosomal circular plasmid inducing Ti tumors A. tumefaciens, using a binary system (WATSON et al., recombinant DNA, Ed. De Boeck University, 273-292,1994).

To do this, two vectors are built. In one of these vectors, the T-DNA region was deleted except for the right and left edges, with a marker gene inserted between them to allow selection in plant cells. The other partner of the binary system is an auxiliary Ti plasmid, a modified plasmid that no longer has T-DNA but still contains the virulence vir genes required for the transformation of the plant cell. This plasmid is maintained in Agrobacterium.

 For the transformation of monocotyledons, in particular maize, the method described by ISHIDA et al. (Nature Biotechnology, 14, 745-750, 1996).

 The genetic transformation of plants by a polynucleotide encoding a mutant CCoAOMT-2 according to the invention makes it possible to obtain transgenic plants, and in particular maize plants, having increased digestibility.

 It is however necessary that the allele present naturally in the plant is inactivated. Inactivation of this allele can be obtained for example by insertion into

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 the CCoAOMT2 gene (preferentially in the coding region) of a T-DNA or any other transposable element.

 The present invention thus more particularly relates to a process for producing transgenic maize having an increased degree of digestibility, characterized in that it comprises the following steps: a) inactivation of the natural allele of the CCoAOMT2 gene of the plant to transform; b) the genetic transformation of the plant resulting from step a) by a polynucleotide encoding a mutant CCoAOMT-2 according to the invention.

 Alternatively, corn having an increased digestibility can be obtained by site-directed mutagenesis, in order to introduce into the natural allele of the CCoAOMT2 gene the modifications necessary for the expression of a mutant CCoAOMT-2 according to the invention.

 Site-directed mutagenesis methods usable in the context of the present invention are known in themselves to those skilled in the art; they are for example described in the book by D. Tagu (eds INRA 1999).

 Advantageously, at the end of the transgenesis or site-directed mutagenesis, the plants containing the gene coding for a mutant CCoAOMT-2 according to the invention may be selected by detection of this gene using one of the detection methods according to the invention. to the invention described above.

 The invention also relates to transformed plants obtained by a method of transgenesis or site-directed mutagenesis according to the invention, as well as the descendants of these plants. The invention also encompasses products obtained from these plants, such as plant organs or tissues, cells, seeds, etc.

 Finally, the invention also relates to derived products, and especially forage, obtained from these plants.

 Alleles of the CCoAOMT2 gene favorable for digestibility, and containing polymorphisms different from those identified in the SEQ ID NO: 3 allele can be

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 identified from high digestibility maize lines, and producing lignins with a higher S / G ratio than lignins from conventional corn silage varieties. The CCoAOMT2 alleles present in these lines can be isolated and sequenced and their sequences compared with that of the wild-type CCoAOMT2 gene, in order to detect the polymorphisms associated with these alleles. Polynucleotides reproducing the sequence of these alleles, as well as mutant CCoAOMT-2 produced by these can be used in all the applications described above.

 The present invention will be better understood with the aid of the additional description which follows, which refers to nonlimiting examples of methods of detecting a CCoAOMT2 gene allele favoring digestibility, and of use of this allele for obtaining transgenic plants.

EXAMPLE 1: DEVELOPMENT OF A DETECTION TEST FOR A POLYMORPHIC MARKER COMPRISING DIGESTIBILITY

 The next 9 maize lines (ranked in order of decreasing digestibility) were tested for the presence of one of the polymorphic markers identified on the sequence SEQ ID NO: 3.

Lines F4>A>B>C>D>E>F> G
The line F4 is that from which the allele SEQ ID NO: 3 was obtained.

Used primers
The marker chosen is the deletion of 9 nucleotides between nucleotides 86 and 87 of the sequence SEQ ID NO: 3, which corresponds to nucleotides 81 to 91 of the sequence SEQ ID NO: 1.

2 pairs of primers have been defined:
The first is intended to allow selective amplification of the allele carrying the desired deletion. If this allele is not present in the sample, no amplification is observed.

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 The second frames the location of the desired deletion. The presence or absence of an allele carrying this deletion is detected by the size of the amplification product.

 The first pair of primers comprises: - a selective sense primer, located astride the location of the deletion. This sequence is represented by the sequence SEQ ID NO: 7; the first 4 bases of this sequence correspond to additional bases added to increase the melting temperature of the primer and thus increase the specificity of the PCR; an antisense, non-selective primer: This primer, located immediately before the stop codon of the gene, is common to the different alleles of the CCoAOMT2 gene. Its sequence is represented under the number SEQ ID NO: 8.

 The second pair of primers comprises a sense primer defined by the sequence SEQ ID NO: 5 and an antisense primer defined by the sequence SEQ ID NO: 6.

 Each of these primer pairs was tested on genetic material obtained from lines E, D, F, G and F4 (30 ng of genomic DNA of each line).

Amplification by the pair of primers SEQ ID NO: 7 and ID NO: 8
The PCR is carried out in a volume of 50 μl of mixture having the following composition:
Matrix: 30 ng genomic DNA primers: 0.2 μM of each dNTPs: 200 μM each
Buffer: 5 μl of 10 X buffer containing 100 mM tris-HCl, 500 mM Kcl, 15 mM MgCl 2 and 0.01% gelatin pH 8.3
Polymerase: REDTaq GENOMIC DNA POLYMERASE (SIGMA). 2.5 units of polymerase are added to the 50 μl of reaction mixture.

 Amplification conditions:

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5 min 95 C 33 cycles :

30s 9SoC J 30 s à 670C 1 min à 72 Oc

5 min à 72 C
Pour obtenir des témoins positifs d'amplification, on utilise le couple d'amorces suivant, qui amplifie le gène CCoAOMT2 pour toutes les lignées, avec une efficacité moindre d'amplification pour la lignée F4, car une partie de l'amorce sens recouvre la zone délétée dans cette lignée) : Amorce sens : GCG ACC GAG GCG ACC AAG ACG (SEQ ID NO : 9) correspondant aux positions 83 à 103 de la séquence SEQ ID NO : 1.

5 min 95 C 33 cycles:

30s 9SoC J 30s at 670C 1 min at 72 Oc

5 min at 72 C
To obtain positive amplification controls, the following pair of primers is used, which amplifies the CCoAOMT2 gene for all the lines, with a lower amplification efficiency for the F4 line, since part of the forward primer covers the zone deleted in this line): sense primer: GCG ACC GAG ACCAG GCAG ACC (SEQ ID NO: 9) corresponding to positions 83 to 103 of the sequence SEQ ID NO: 1.

Antisense Primer: CTT GAC GCG GCG GCA GAG CGT GAC GCC GTC GC (SEQ ID NO: 8)
25 μl of the product of the PCR reaction are deposited on a mini agarose gel (1%) supplemented with ethidium bromide.

The migration is performed at 100 volts for 15 to 20 minutes.

The results are illustrated in Figure 3:
The first 5 depots correspond to the amplifications obtained with the pair of primers SEQ ID NO: 7 and SEQ ID NO: 8 on the lines (from left to right) G (lane 1), F (lane 2), E (lane 3 ), D (lane 4) and F4 (lane 5).

 The central repository (lane 6) corresponds to the 1 kb molecular size marker.

 The last 5 deposits correspond to the positive controls, obtained with the control primer pair SEQ ID NO: 9 and SEQ ID NO: 8: lines G (lane 7), F (lane 8), E (lane 9), D ( lane 10) and F4 (lane 11).

 The F4 line is characterized by a net band of about 1 kb, corresponding to the amplification product obtained with the primers SEQ ID NO: 7 and SEQ ID NO: 8. For the other lines, and with these same primers either no band appears at this level, ie the bands have a very low intensity.

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 Other experiments were carried out using a primer hybridization temperature ranging from 66 ° C. to 68 ° C. The same amplification specificity was observed for the pair of primers SEQ ID NO: 7 and SEQ ID NO: 8 .

Amplification by the pair of primers SEQ ID NO: 5 and SEQ ID NO: 6
The pair of primers defined by the sequences SEQ ID NO: 5 and SEQ ID NO: 6 was tested on lines E, D, F, G and F4.

 The reaction medium is the same as that used above but the reaction volume is doubled to 100 μl in order to deposit 70 μl of the PCR product on a 4% agarose-metaphoreal gel (TEBU) supplemented with ethidium bromide. This gel has a high resolution capacity, making it possible to demonstrate a difference in size of 4 bp for DNA fragments whose size is between 200 and 16 bp.

Cycle: 5 min 95 C 30 cycles: 30 s 950C
30s to 650C
30 s at 72 C 5 min at 720C
The migration takes place for 4 hours at 90 V.

 The results are shown in Figure 4.

 The first deposition (lane 1) corresponds to the 100 bp molecular size marker.

 The following 5 deposits correspond to the amplification products of lines D (lane 2), G (lane 3), F (lane 4), E (lane 5), F4 (lane 6).

 These results show that the amplifiat obtained from the F4 line is smaller than that obtained from the other lines. The pair of primers used is therefore very distinctive of the desired polymorphism.

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EXAMPLE 2 Transformation of Plants by an Alle1 of the CCOAOMT2 Gene Which Is Favorable for DIGESTIBILITY
The maize F4 line, which has a high digestibility, was used as starting material.

 The CCoAOMT2 gene was obtained from cDNA extracted from this line by PCR amplification using the primers SEQ ID NO: 8 and SEQ ID NO: 9.

 The cloning of the PCR fragments obtained corresponding to the coding region of CCoAOMT-2 was carried out using the PGEM-T EASY vector system according to the supplier's instructions (PROMEGA).

 The PCR fragment is ligated into the pGEM-T EASY vector (PROMEGA). The competent E. coli JM109 bacteria (marketed by PROMEGA) are transformed using this construct.

Transformation of plant cells by Agrobacterium tumefaciens
The transformation technique described by ISHIDA et al. (1996, supra) is used from immature embryos 10 days after fertilization. All media used are described in this publication.

 A superbinary vector is constructed by homologous recombination between an intermediate vector (pBS11 or pBS12) carrying a resistance marker, and in which the excised mutant CCoAOMT2 gene of the pGEM-T EASY vector, and the pSB1 vector of JAPAN TOBACCO ( EP 672 752) which contains: virB and virG genes of plasmid pTiBo542 present in the supervirulent strain A281 of Agrobacterium tumefaciens (ATCC 37349). The vectors pBS11, pBS12, and pSB1 are described in Application EP 672 752 in the name of JAPAN TOBACCO.

 The transformation is carried out by contacting the immature embryos of the maize plants for at least 5 minutes with Agrobacterium tumefaciens LBA 4404 containing the superbinary vector.

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 The embryos are then placed on LSA medium for 3 days in the dark and at 25 C. A first selection is performed on the transformed calli: the embryogenic calli are transferred to LSD5 medium containing phosphinotricin at 5 mg / l and the cefotaxime at 250 mg / l (elimination or limitation of contamination with Agrobacterium tumefaciens). This step 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 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 before. The third selection step is to excise the type I callus (fragments of 1 to 2 mm) and transfer them 3 weeks in the dark and 250C on LSD 10 medium in the presence of cefotaxime.

 Regeneration of the seedlings is performed by excising the proliferating type I calli and transferring them to LSZ medium in the presence of 5 mg / l phosphinotricin and cefotaxime for 2 weeks at 22 ° C. under continuous light.

 Regenerated seedlings are transferred to RM + G2 medium containing 100 mg / l AUGMENTIN &commat; for 2 weeks at 22 C and under continuous illumination for the development stage. The resulting plants are then transferred to the phytotron for acclimation.

Transformation of plant cells by bombardment
Calli aged 2 months, obtained after the culture of immature embryos on a callogenesis medium are used for the transformation.

 A plasmolysing treatment makes it possible to reduce the volume of the vacuole and thus limits the bursting of cells during the firing (pretreatment of 4 hours and a post-treatment of 16 hours on a medium Sorbitol 0.2M and Mannitol 0.2 M).

 The plant material is homogeneously distributed over all of the boxes.

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 15 mg of sterile tungsten microbeads are mixed in 150 μl of sterilized water by microfiltration.

 The mutant CCoAOMT2 gene is inserted into a plasmid pDM302 carrying a selection marker.

d'intérêt (à 1 pg/pl),
2,5 pl d'ADN (à 1 pg/pl) du plasmide portant le gène CCoAOMT2 et le marqueur de sélection,
25 pl de CaClzà 2,5 M,
10 pi de spermidine à 0,1 M. For 5 shots, the following mixture is prepared:
25 μl of tungsten microbeads,
2.5 μl of plasmid DNA carrying the gene

of interest (at 1 pg / pl),
2.5 μl of DNA (at 1 μg / μl) of the plasmid carrying the CCoAOMT2 gene and the selection marker,
25 μl of CaCl 2 at 2.5 M,
10 μl of 0.1M spermidine

 This mixture is allowed to settle in ice for at least 5 minutes.

 Five Petri dishes containing the material to be bombarded are placed on 15 g / L agar plates.

 50 μl of supernatant from the decanted tube are removed and the remaining mixture is vortexed, then 2 μl of it is deposited on the grid of the sterile syringe, which is screwed onto the support in the barrel.

A sterile gauze filter is placed on the petri dish which is placed 19 cm into the barrel enclosure. The vacuum is pushed inside the chamber at a pressure of 25 mbar
The shot is fired. The pressure of the helium in the barrel is 8 bars.

 The bombarded plant material is returned to its original Petri dish (Mannitol / Sorbitol). The dishes are placed at 26 ° C. and in the dark for 16 hours for post-bombardment plasmolysing treatment (mannitol / sorbitol).

 The selection pressure is applied 16 hours after firing and is maintained at the same concentration of the selective agent (BIALAPHOS 5 mg / L) during all regeneration steps. The seedlings are then transplanted into potting soil (small pots) for 1 week and then transferred into 20-liter pots (peat-puddolane mixture) in the transgenic greenhouse or in an air-conditioned chamber (Temperature 240C day / 20 C night, photoperiod 16h day /

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 8 pm night, humidity 80%). The plants are self-fertilized or crossed.

Claims (31)

1) Method for evaluating the digestibility of a forage plant, characterized in that it comprises detecting the presence or absence, in biological material from said plant, of an allele favorable to digestibility of the CCoAOMT2 gene.  2) Method according to claim 1, characterized in that said forage plant is maize.  3) Process according to any one of claims 1 or 2, characterized in that said allele favorable for digestibility codes for a mutant CCoAOMT-2 protein having compared to the wild-type CCoAOMT-2 protein of sequence SEQ ID NO: 2, at least one of the following mutations: a) the deletion of the Glu-Ala-Thr peptide sequence, located between positions 6 and 10 of the sequence SEQ ID NO: 2; b) inserting the Asn-Gly-Asn-Gly-Asn-Gly peptide sequence between positions 26 and 27 of the sequence SEQ ID NO: 2; c) substituting the His residue at position 37 of the sequence SEQ ID NO: 2 by an Arg residue; d) the substitution of the residue Asn at position 188 of the sequence SEQ ID NO: 2 by a residue Ser.  4) Method according to claim 3, characterized in that said mutant CCoAOMT-2 comprises at least mutation a).  5) Method according to any one of claims 1 to 4, characterized in that the detection of the presence of a CCoAOMT2 gene allele favorable to digestibility is performed by detecting the form associated with said allele of at least one polymorphism of said gene.  6) Method according to claim 5, characterized in that said polymorphism is chosen from: a 5 base pair insertion, of ACTGC sequence, between nucleotides 55 and 56 of the reference sequence SEQ ID NO: 1; <Desc / Clms Page number 30> 692 of the reference sequence SEQ ID NO: 1; a substitution A -) - C at position 667 of the reference sequence SEQ ID NO: 1; a substitution C - + G at position 698 of the reference sequence SEQ ID NO: 1; a TG # CC substitution at positions 664-665 of the reference sequence SEQ ID NO: 1; an A-G substitution at position 904 of the reference sequence SEQ ID NO: 1. 668 and 672 of the reference sequence SEQ ID NO: 1; at least one C # T substitution at position 635 or 648 of the reference sequence SEQ ID NO: 1; at least one T-C substitution in position 638,672 or CTCTCTGTTGCTCGTCCC, between nucleotides 703 and 704 of the reference sequence SEQ ID NO: 1; a deletion of 3 base pairs between the nucleotides TGCCCCTTTTCTCTCT, between nucleotides 631 and 632 of the reference sequence SEQ ID NO: 1; - an insertion of 18 base pairs, of sequence ID NO: 1; a C-G substitution at position 178 of the reference sequence SEQ ID NO: 1; a substitution A-G at position 180 of the reference sequence SEQ ID NO: 1; a Go A substitution at position 227 of the reference sequence SEQ ID NO: 1; an A-G substitution at position 237 of the reference sequence SEQ ID NO: 1; a CG- + TT substitution at positions 259-260 of the reference sequence SEQ ID NO: 1; an A- + G substitution at position 466 of the reference sequence SEQ ID NO: 1; - an insertion of 15 base pairs, of sequence 81 and 91 of the reference sequence SEQ ID NO: 1; an insertion of 18 base pairs between nucleotides 129 and 130 of the SEQ reference sequence  a deletion of 9 base pairs between the nucleotides <Desc / Clms Page number 31>  7) Method according to claim 6, characterized in that said polymorphism is a deletion of 9 base pairs between nucleotides 81 and 91 of the reference sequence SEQ ID NO: 1.  8) Method according to claim 6, characterized in that said polymorphism is an 18-base pair insertion, sequence CAACGGCAACGGCAACGG, between nucleotides 129 and 130 of the reference sequence SEQ ID NO: 1.  9) Process according to any one of claims 5 to 8, characterized in that the detection of said polymorphism comprises the selective hybridization, with genetic material from said plant, of at least one polynucleotide probe specific for the form of said polymorphism present in the desired CCoAOMT2 allele.  10) Method according to any one of claims 5 to 9, characterized in that it comprises the amplification, from genetic material from said plant, of the CCoAOMT2 gene or a region thereof comprising at least one polymorphism to detect, and determining the form of said polymorphism present in said plant.  11) Method according to claim 10, characterized in that the form of said polymorphism present in said plant is determined by the size of the amplification product.  12) Process according to any one of claims 1 to 4, characterized in that the detection of the presence or absence of a gene allele CCoAOMT2 conducive to digestibility is by detection of a mutant CCoAOMT-2 protein presenting, with respect to the wild-type CCoAOMT-2 protein of sequence SEQ ID NO: 2, at least one of the mutations defined in claim 3.  13) A mutant 3-methyl-2-transferablecarbonoyl Coenzyme A (CCoAOMT-2), characterized in that it differs from CCoAOMT-2 of sequence SEQ ID NO: 2 by at least one of the following mutations: <Desc / Clms Page number 32>  a) the deletion of the Glu-Ala-Thr peptide sequence between positions 6 and 10 of the sequence SEQ ID NO: 2; b) inserting the Asn-GlyAsn-Gly-Asn-Gly peptide sequence between positions 26 and 27 of the sequence SEQ ID NO: 2; c) substituting the His residue at position 37 of the sequence SEQ ID NO: 2 by an Arg residue; d) the substitution of the residue Asn at position 188 of the sequence SEQ ID NO: 2 by a residue Ser.  14) CCoAOMT-2 mutant according to claim 13 characterized in that it comprises at least the mutation a).  15) CCoAOMT-2 mutant according to any one of claims 13 or 14, characterized in that it corresponds to the sequence SEQ ID NO: 4.  16) A polynucleotide encoding a mutant CCoAOMT-2 according to any one of claims 13 to 15.  17) A polynucleotide according to claim 16, characterized in that its sequence is selected from; the genomic DNA sequence represented in the list of sequences in the appendix under the number SEQ ID NO: 3; the cDNA sequence derived from said genomic sequence.  18) Polynucleotide chosen from: a fragment of more than 10 bp of a polynucleotide of sequence SEQ ID NO: 3, said fragment comprising at least one of the polymorphisms defined in claim 6; the complementary of said fragment.  19) polynucleotide probe for carrying out a method according to claim 9, characterized in that it comprises a polynucleotide of sequence SEQ ID NO: 3, or its complement, or a polynucleotide according to claim 18.  20) primer pair for carrying out a method according to claim 10, characterized in that it comprises at least one primer comprising a polynucleotide according to claim 18. <Desc / Clms Page number 33>  21) primer pair according to claim 20, characterized in that it is selected from: a pair of primers consisting of the oligonucleotides of sequence SEQ ID NO: 7 and SEQ ID NO: 8. a pair of primers constituted by the oligonucleotides SEQ ID NO: 5 and SEQ ID NO: 6.  22) Use of a polynucleotide according to claim 18 for the detection of a CCoAOMT2 gene allele of but favorable for digestibility.  23) An antibody selectively recognizing a mutant CCoAOMT-2 protein according to any one of claims 13 to 15, or a fragment thereof comprising at least one of mutations a) to d) defined in claim 13.  24) A recombinant expression cassette comprising a polynucleotide according to any of claims 16 to 18.  25) Recombinant vector resulting from the insertion of a polynucleotide according to any of claims 16 to 18, or an expression cassette according to claim 24, into a host vector.  26) A process for producing a recombinant polypeptide, characterized in that it comprises transforming a host cell, prokaryotic or eukaryotic, with a polynucleotide encoding a mutant CCoAOMT-2 according to any one of claims 13 to 15, or for a fragment thereof comprising at least one of mutations a) to d), and recovering said mutant CCoAOMT-2 or fragment produced by said cell.  27) Cell genetically transformed with a polynucleotide according to any one of claims 16 to 18.  28) Cell according to claim 27, characterized in that it is a plant cell.  29) A process for producing a transgenic forage plant having an increased digestibility rate, characterized in that it comprises the following steps: <Desc / Clms Page number 34>  a) the inactivation of the natural allele of the CCoAOMT2 gene of the plant to be transformed; b) the genetic transformation of the plant resulting from step a) by a polynucleotide according to any of claims 16 to 17.  30) A process for producing a forage plant having an increased digestibility level, characterized in that it comprises site-directed mutagenesis of the CCoAOMT2 gene allele present in the plant to be transformed in order to obtain an allele of the CCoAOMT2 gene having the sequence a polynucleotide according to any one of claims 16 to 17.  31) Plant genetically transformed with a polynucleotide according to any one of claims 16 to 17.