AU749274B2 - Methods for obtaining plant varieties - Google Patents

Description

PCT/EP8/6977 *rnf~ fl1 SiAfl Methods for Obtaining Plant Varieties TECHNICAL FIELD The present invention relates to nucleotide sequences which encode polypeptides involved in the DNA mismatch repair systems of plants, and to the polypeptides encoded by those nucleotide sequences. The invention also relates to nucleotide sequences and polypeptide sequences for use in altering the DNA mismatch repair system in plants. The invention also relates to a process for altering the DNA mismatch repair system of a plant cell, to a process for increasing genetic variations in plants and to processes for obtaining plants having a desired characteristic.

BACKGROUND OF THE INVENTION Plant breeding essentially relies on and makes use of genetic variation which occurs naturally within and between members of a family, a genus, a species or a subspecies.

Another source of genetic variation is the introduction of genes from other organisms which may or may not be related to the host plant.

Allelic loci or non-allelic genes which constitute or contribute to desired quantitative growth performance, yield, etc.) or qualitative deposition, content and composition of seed storage products; pathogen resistance genes: etc.) traits that are absent, incomplete or inefficient in a species or subspecies of interest are typically introduced by the plant breeder from other species or subspecies, or de novo. This introduction is often done by crossing, provided that the species to be crossed are sexually compatible. Other means of introducing genomes, individual chromosomes or genes into plant cells or plants are well known in the art. They include cell fusion, chemically aided transfection (Schocher et al., 1986, Biotechnology 4: 1093) and ballistic (McCabe et al., 1988, Biotechnology 6: 923), microinjection (Neuhaus et al., 1987, TAG 75: electroporation of protoplasts (Chupeau et al., 1989, Biotechnology 7: 53) or microbial transformation methods such as Agrobacterium mediated transformation (Horsch et al., 1985, Science 227: 1229; Hiei et al., 1996, Biotechnology 14: 745).

However, when a foreign genome, chromosome or gene is introduced into a plant, it will often segregate in subsequent generations from the genome of the recipient plant or plant cell during mitotic and meiotic cell divisions and, in consequence, become lost from the host plant or plant cell into which it had been introduced. Occasionally, however, the introduced genome, chromosome or gene physically combines entirely or in part with the genome. chromosome or gene of the host plant or plant cell in a process which is called recombination.

Recombination involves the exchange of covalent linkages between DNA molecules in regions of identical or similar sequence. It is referred to here as homologous recombination if donor and recipient DNA are identical or nearly identical (at least 99% WA 00/1 4092 PCT/EP98/06977 vI 001 A J 2 base sequence identity), and as homeologous recombination if donor and recipient DNA are not identical but are similar (less than 99% base sequence identity).

The ability of two genomes, chromosomes or genes to recombine is known to depend largely on the evolutionary relation between them and thus on the degree of sequence similarity between the two DNA molecules. Whereas homologous recombination is frequently observed during mitosis and meiosis, homeologous recombination is rarely or never seen.

From a breeder's perspective, the limits within which homologous recombination occurs, therefore, define a genetic barrier between species, varieties or lines, in contrast o1 to homeologous recombination which can break this barrier. Homeologous recombination is thus of great importance for plant breeding. Accordingly there is a need for a process for enhancing the frequency of homeologous recombination in plants. In particular, there is a need for a process of increasing homeologous recombination to significantly shorten the length of breeding programs by reducing the number of crosses required to obtain an otherwise rare recombination event.

At least in Escherichia coli. homologous and homeologous recombination are known to share a common pathway that requires among others the proteins RecA, RecB, RecC.

RecD and makes use of the SOS induced RuvA and RuvB, respectively. It has been suggested that mating induced recombination follows the Double-Strand Break Repair model (Szostak et al., 1983, Cell 33, 25-35), which is widely used to describe genetic recombination in eukaryotes. Following the alignment of homologous or homeologous DNA double helices the RecA protein mediates an exchange of a single DNA strand from the donor helix to the aligned recipient DNA helix. The incoming strand screens the recipient helix for sequence complementarity, seeking to form a heteroduplex by hydrogen bonding the complementary strand. The displaced homologous or homeologous strand of the recipient helix is guided into the donor helix where it base pairs with its counterpart strand to form a second heteroduplex. The resulting branch point then migrates along the aligned chromosomes thereby elongating and thus stabilising the initial heteroduplexes.

Single stranded gaps (if present) are closed by DNA synthesis. The strand cross overs (Holliday junction) are eventually resolved enzymatically to yield the recombination products.

Although in wild type E. coli homologous and homeologous recombination are thus mechanistically similar if not identical, homologous recombination in conjugational crosses E. coli x E. coli occurs five orders of magnitude more frequently than homeologous recombination in conjugational crosses E. coli x S. typhimurium (Matic et al. 1995; Cell 80, 507-515). The imbalance in favour of homologous recombination was shown to be caused largely by the bacterial MisMatch Repair (MMR) system since its wn oo/I 014 PCT/EP98/06977 3 inactivation increased the frequency of homeologous recombination in E. coli up to 1000 fold (Rayssiguier et al. 1989. Nature 342. 396-401).

In E. coli. the MMR system (reviewed by Modrich 1991. Annual Rev Genetics 229-253) is composed of only three proteins known as MutS, MutL and MutH. MutS recognizes and binds to base pair mismatches. MutL then forms a stable complex with mismatch bound MutS. This protein complex now activates the MutH intrinsic single stranded endonuclease which nicks the strand containing the misplaced base and thereby prepares the template for DNA repair enzymes.

During recombination. MMR components inhibit homeologous recombination. In to vitro experiments demonstrated that MutS in complex with MutL binds to mismatches at the recombination branch point and physically blocks RecA mediated strand exchange and heteroduplex formation (Worth et al., 1994; PNAS 91, 3238-3241). Interestingly, the SOS dependent RuvAB mediated branch migration is insensitive to MutS/MutL, explaining the observed slight increase in SOS dependent homeologous recombination.

Homeologous mating even induces the SOS response, thereby taking advantage of RuvAB induction (Matic et al. 1995, Cell 80. 507-515).

The MMR system thus appears to be a genetic guardian over genome stability in E.

coli. In this role it essentially determines the extent to which genetic isolation, that is, speciation, occurs. The diminished sensitivity of the SOS system to MMR, however, allows (within limits) for rapid genomic changes at times of stress, providing the means for fast adaptation to altered environmental conditions and thus contributing to intraspecies genetic variation and species evolution.

The important role of MMR in preserving genomic integrity has been established also in certain eukaryotes. In its efficiency, the human MMR, for example, may even counteract potential gene therapy tools such as triple-helix forming oligonucleotides including RNA-DNA hybrid molecules (Havre et al., 1993, J. Virology 67: 7234-7331; Wang et al., 1995, Mol. Cell. Biol. 15: 1759-1768; Kotani et al., 1996, Mol. Gen.

Genetics 250: 626-634; Cole-Strauss et al., 1996, Science 273: 1387-1389). Such oligonucleotides are designed to introduce single base changes into selected DNA target sequences in order to inactivate for example cancer genes or to restore their normal function. The resulting base mismatches however are recognised by the mismatch repair system which then directs removal of the mismatched base, thereby reducing the efficiency of oligonucleotide induced site-specific mutagenesis.

To date, two families of related genes, homologous to the bacterial MutS and MutL genes have been identified or isolated in yeast and mammals (recent reviews by Arnheim and Shibata, 1997, Curr. Opinion Genet. Dev. 7, 364-370; Modrich and Lahue, 1996, Annual Rev. Biochem. 65, 101-133; Umar and Kunkel, 1996, Eur. J. Biochem. 238, 297- 307). Biochemical and genetic analysis indicated that eukaryotic MutS homologs (MSH) and MutL homologs (MLH, PMS), respectively, fulfil similar protein functions as their bacterial counterparts. Their relative abundance, however, could reflect different mismatch specificity and/or specialisation for different tissues or organelles or developmental processes such as mitotic versus meiotic recombination.

To date, six different genes homologous to MutS have been isolated in yeast (yMSH), and their homologs have been found in mouse (mMSH) and human (hMSH), respectively.

Encoded proteins yMSH2, yMSH3 and yMSH6 appear to be the main MutS homologs involved in MMR during mitosis and meiosis in yeast, where the complementary proteins MSH3 and MSH6 alternatively associate with MSH2 to recognise different mismatch substrates (Masischky et al., 1996, Genes Dev. 10, 407-420). Similar protein interactions have been demonstrated for the human homologs hMSH2, hMSH3 and hMSH6 (Acharya et al., 1996, PNAS 93, 13629-13634).

MutL homologs (MLH and PMS), recently reviewed by Modrich and Lahue (1996, Annual Rev. Biochem. 65, 101-133) have so far been found in yeast (yMLH1 and yPMS1), 15 mouse (mPMS2) and human (hMLHI, hPMS1 and hPMS2). The hPMS2 is a member of a family of at least 7 genes (Horii et al., 1994, Biochem. Biophys. Res. Commun. 204, 1257- 1264) and its gene product is most closely related to yPMS1. Prolla et al. (1994, Science 265, 1091-1093) presented evidence for yPMS1 and yMLH1 to physically associate with each other and, together, to interact with the MutS homolog yMSH2 to form a ternary complex involved in mismatch substrate binding.

However, while medical interest in mismatch repair has prompted extensive research on MMR in bacteria, yeast and mammals, MMR genes have not been isolated from higher plants prior to the present invention and no attempts to adjust the plant MMR to plant breeding needs have been reported.

SUMMARY OF THE INVENTION Herein disclosed is an isolated and purified DNA molecule comprising a polynucleotide sequence encoding a polypeptide functionally involved in the DNA mismatch repair system of a plant. Also disclosed is an isolated and purified DNA molecule comprising a polynucleotide sequence encoding a polypeptide which is homologous to a mismatch repair polypeptide of a yeast or of a human. More particularly, the polynucleotide sequences encode polypeptides which are homologous to the mismatch repair polypeptides MSH3 and MSH6 of Saccharomyces cerevisiae. Still more particularly, herein disclosed are coding sequences of the genes AtMSH3 and AtMSH6 of Arabidopsis thaliana, as defined hereinbelow, and polynucleotide sequences encoding polypeptides which are homologous to polypeptides encoded by AtMSH3 and AtMSH6.

Thus, according to a first embodiment of the invention, there is provided an isolated and purified DNA polynucleotide comprising a nucleotide sequence encoding a plant polypeptide functionally involved in the DNA mismatch repair system of said plant, wherein said polypeptide is homologous to AtMSH3 (SEQ ID NO: 19) or to AtMSH6 (SEQ ID NO: 31).

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LbV 0 In a related aspect of the first embodiment, there is provided an isolated and purified DNA polynucleotide comprising a nucleotide sequence encoding a DNA mismatch repair protein active in plants, or encoding a fragment of said protein having the biological activity thereof, and wherein said protein or fragment thereof shares at least 50% identity with the amino acid sequence ofAtMSH3 (SEQ ID NO: 19) or AtMSH6 (SEQ ID NO: 31).

In another related aspect of the first embodiment, there is provided an isolated and purified DNA polynucleotide comprising a nucleotide sequence encoding a DNA mismatch repair protein active in plants, or encoding a fragment of said protein having the biological activity thereof, and wherein said nucleotide sequence shares at least 50% identity with the nucleotide sequence ofAtMSH3 (SEQ ID NO: 18) or AtMSH6 (SEQ ID NO: Also herein disclosed is an isolated and purified polypeptide functionally involved in the DNA mismatch repair system of a plant, for example a polypeptide which is homologous to a mismatch repair polypeptide of a yeast or of a human such as a polypeptide encoded by the genes AtMSH3 or AtMSH6 ofArabidopsis thaliana, as defined hereinbelow.

According to a second embodiment of the invention, there is provided an isolated and purified plant polypeptide functionally involved in the DNA mismatch repair system of said plant, wherein said polypeptide is homologous to AtMSH3 (SEQ ID NO: 19) or to AtMSH6 (SEQ ID NO: 31).

In a related aspect of the second embodiment, there is provided an isolated and purified plant polypeptide functionally involved in the DNA mismatch repair system of said plant, selected from the group consisting of a polypeptide encoded by the gene AtMSH3 (SEQ ID NO: 18), a polypeptide encoded by the gene AtMSH6 (SEQ ID polypeptides homologous to a polypeptide encoded by the gene AtMSH3 (SEQ ID NO: 18) and polypeptides homologous to a polypeptide encoded by the gene AtMSH6 (SEQ ID 25 In another related aspect of the second embodiment, there is provided an isolated and purified polypeptide encoded by the gene AtMSH3 (SEQ ID NO: 18) or by the gene AtMSH6 (SEQ ID In another related aspect of the second embodiment, there is provided an isolated and purified DNA mismatch repair protein active in plants, or a fragment of said protein having the biological activity thereof, wherein said protein or fragment thereof shares at least identity with the amino acid sequence of AtMSH3 (SEQ ID NO: 19) or AtMSH6 (SEQ ID NO: 31).

In another related aspect of the second embodiment, there is provided an isolated and 35 purified DNA mismatch repair protein active in plants, or a fragment of said protein having the biological activity thereof, wherein said protein, or fragment thereof is encoded by a nucleotide sequence which shares at least 50% identity with the nucleotide sequence of AtMSH3 (SEQ ID NO: 18) or AtMSH6 (SEQ ID NO: Also disclosed herein is an isolated and purified DNA molecule comprising a polynucleotide sequence selected from the group consisting of a sequence encoding a polynucleotide which is capable of interfering with the expression of a plant polynucleotide sequence encoding a polypeptide which is homologous to a mismatch repair polypeptide of a yeast or of a human and thereby disabling said plant polynucleotide sequence; and (ii) a sequence encoding a polypeptide capable of disrupting the DNA mismatch repair system of a plant.

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A395498auspcci Thus, according to a third embodiment of the invention, there is provided an isolated and purified DNA polynucleotide comprising a nucleotide sequence selected from the group consisting of: a sequence encoding a polynucleotide which is capable of interfering with the expression of a plant polynucleotide sequence encoding a polypeptide which is homologous to a mismatch repair polypeptide of a yeast or of a human and thereby disabling said plant polynucleotide sequence; and (ii) a sequence encoding a plant polypeptide capable of disrupting the DNA mismatch repair system of a plant; wherein said polypeptide is homologous to AtMSH3 (SEQ ID NO: 19) or to AtMSH6 (SEQ ID NO: 31).

Also herein disclosed is a chimeric gene comprising a DNA sequence selected from the group consisting of a sequence encoding a polynucleotide which is capable of interfering with the expression of a plant polynucleotide sequence encoding a polypeptide which is homologous to a mismatch repair polypeptide of a yeast or of a human and thereby disabling said plant polynucleotide sequence, and (ii) a sequence encoding a polypeptide capable of disrupting the DNA mismatch repair system of a plant; together with at least one regulation element capable of functioning in a plant cell. Examples of such regulation elements include constitutive, inducible, tissue type specific and cell type specific promoters such as 35S, NOS, PRIa, AoPR1 and DMC1. Typically, a chimeric gene of the fourth embodiment will also include at least one terminator sequence, more typically exactly one terminator sequence.

According to a fourth embodiment of the invention, there is provided a chimeric gene comprising: a DNA polynucleotide according to the eighth embodiment; and at least one regulation element capable of functioning in a plant cell.

In a related aspect of the fourth embodiment, there is provided a recombinant DNA construct comprising a DNA polynucleotide according to the eighth embodiment and a regulation element capable of causing overexpression of said polypeptide in a cell of said plant.

In the third and fourth embodiments, said interference, by said polynucleotide sequence, with the expression of a plant polynucleotide sequence encoding a polypeptide which is homologous to a mismatch repair peptide of a yeast or a human typically occurs by hybridisation or by co-suppression.

According to a fifth embodiment of the invention there is provided a plasmid or vector comprising a chimeric gene of the fourth embodiment. A vector of the fifth embodiment may be, for example, a viral vector or a bacterial vector.

According to a sixth embodiment of the invention, there is provided a plant cell stably transformed, transfected or electroporated with a plasmid or vector of the fifth embodiment.

According to a seventh embodiment of the invention, there is provided a plant comprising a cell of the sixth embodiment.

According to an eighth embodiment of the invention, there is provided a process for at least partially inactivating a DNA mismatch repair system of a plant cell, comprising transforming or transfecting said plant cell with a DNA polynucleotide of the first or third embodiments or a chimeric gene of the fourth embodiment or a plasmid or vector of the fifth embodiment, and causing said nucleotide sequence to express said polynucleotide or said polypeptide. Plant cells in which the DNA mismatch repair system has been at least A395498auspeci partially inactivated by said process are also provided, as are plants derived therefrom. In related aspects to the eighth embodiment, there is also provided a DNA polynucleotide according to the first or third embodiments, when used for at least partially inactivating a DNA mismatch repair system of a plant cell.

Also herein disclosed is a process for increasing genetic variation in a plant comprising obtaining a hybrid plant from a first plant and a second plant, or cells thereof, said first and second plants being genetically different; altering the mismatch repair system in said hybrid plant; permitting said hybrid plant to self-fertilise and produce offspring plants; and screening said offspring plants for plants in which homeologous recombination has occurred. For example, homeologous recombination may be evidenced by new genetic linkage of a desired characteristic trait or of a gene which contributes to a desired characteristic trait.

Thus, according to a ninth embodiment of the invention, there is provided a process for increasing genetic variation in a plant comprising obtaining a hybrid plant from a first plant and a second plant, or cells thereof, said first and second plants being genetically different; altering the mismatch repair system in said hybrid plant by altering the expression i of an MSH3 gene and/or an MSH6 gene of said plant by introducing into said plant, or one or more cells thereof, a DNA polynucleotide according to the first or third embodiments, or a recombinant DNA construct, chimeric gene or plasmid or vector comprising said DNA 20 polynucleotide; permitting said hybrid plant to self-fertilise and produce offspring plants; and screening said offspring plants for plants in which homeologous recombination has occurred.

In a modified version of the ninth embodiment, there is provided a process for increasing genetic variation in a plant comprising obtaining a hybrid plant from a first plant and a second plant, or cells thereof, said first and second plants being genetically different; wherein the expression of an MSH3 gene is altered in said first plant and the expression of an MSH6 gene is altered in said second plant, said gene expression alterations being introduced into said plants, or one or more cells thereof, by inserting into said plants a DNA polynucleotide according to the first or third embodiments, or a recombinant DNA 30 construct, chimeric gene or plasmid or vector comprising said DNA polynucleotide; permitting said hybrid plant to self-fertilise and produce offspring plants; and screening said offspring plants for plants in which homeologous recombination has occurred.

In related aspects to the ninth embodiment, there is also provided a DNA polynucleotide according to the first or third embodiments, when used for increasing genetic variation in a plant.

Plants comprising one or more cells in which the DNA mismatch repair system has been altered, and optionally restored, obtained by the process of the ninth embodiment are also provided.

Also herein disclosed is a process for obtaining a plant having a desired characteristic, comprising altering the mismatch repair system in a plant, cell or plurality of cells of a plant which does not have said desired characteristic, permitting mutations to persist in said cells to produce mutated plant cells, deriving plants from said mutated plant cells, and screening said plants for a plant having said desired characteristic.

o r Thus, according to a tenth embodiment of the invention, there is provided a process for obtaining a plant having a desired characteristic, comprising altering the mismatch repair A395498auspeci system in a plant, cell or plurality of cells of a plant which does not have said desired characteristic, permitting mutations to persist in said cells to produce mutated plant cells, deriving plants from said mutated plant cells, and screening said plants for a plant having said desired characteristic, wherein said mismatch repair system is altered by altering the expression of an MSH3 gene and/or an MSH6 gene of said plant by introducing into said plant, cell or plurality of cells, a DNA polynucleotide according to the first or third embodiments, or a recombinant DNA construct, chimeric gene or plasmid or vector comprising said DNA polynucleotide.

In related aspects to the tenth embodiment, there is also provided a DNA polynucleotide according to the first or third embodiments, when used for obtaining a plant having a desired characteristic.

Plants having desired characteristics and comprising one or more cells in which the DNA mismatch repair system has been altered, and optionally restored, obtained by the process of the tenth embodiment are also provided.

In a preferred form of the ninth and tenth embodiments of the invention, the step of altering the mismatch repair system comprises introducing into said hybrid plant, plant, cell or cells a chimeric gene of the fourth embodiment and permitting the chimeric gene to express a polynucleotide which is capable of interfering with the expression of a plant polynucleotide sequence in a mismatch repair gene of the hybrid plant, plant, cell or cells, or 20 a polypeptide capable of disrupting the DNA mismatch repair system of the hybrid plant or cells.

Also herein disclosed are: an oligonucleotide capable of hybridising at 45 0 C under standard PCR conditions to a DNA molecule of the first embodiment; an oligonucleotide capable of hybridising at 60 0 C under standard PCR conditions to the DNA of SEQ ID NO: 25 18 and an oligonucleotide capable of hybridising at 45 0 C under standard PCR conditions to the DNA of SEQ ID Thus, according to an eleventh embodiment of the invention, there is provided an oligonucleotide capable of hybridising at 60 0 C under standard PCR conditions to the DNA of SEQ ID NO: 18 with the proviso that said oligonucleotide is other than SEQ ID NO:1 or SEQ ID NO:2.

According to a twelfth embodiment of the invention, there is provided an oligonucleotide capable of hybridising at 45 0 C under standard PCR conditions to the DNA of SEQ ID NO:30 with the proviso that said oligonucleotide is other than SEQ ID NO: 1 or SEQ ID NO:2.

According to a thirteenth embodiment of the invention, there is provided an oligonucleotide according to the eleventh embodiment, when used for detecting or isolating a plant MSH3 homolog.

According to a fourteenth embodiment of the invention, there is provided an oligonucleotide according to the twelfth embodiment, when used for detecting or isolating a plant MSH6 homolog.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 provides a diagrammatic representation of the primer sequences used to isolate AtMSH3.

Figure 2 is a plasmid map of clone 52, showing restriction enzyme cleavage sites in T 45 the 5' half of the full-length cDNA for AtMSH3.

A395498auspeci xdU\ 00o/1AOQ 9 PCT/EP98/06977 Figure 3 is a plasmid map of clone 13. showing restriction enzyme cleavage sites in the 3' halt of the full-length cDNA for AtMSH3.

Figure 4 is a sequence listing of the coding sequence of AtMSH3, together with a deduced sequence of the encoded polypeptide.

Figure 5 is a protein alignment of yeast (Saccharomyces cerevisiae) and Arabidopsis thaliana MSH3 protein.

Figure 6 provides a diagrammatic representation of the primer sequences used to isolate AtMSH6.

Figure 7 is a plasmid map of clone 43, showing restriction enzyme cleavage sites in o1 the 5' half of the full-length cDNA for AtMSH6.

Figure 8 is a plasmid map of clone 62, showing restriction enzyme cleavage sites in the 3' half of the full-length cDNA for AtMSH6.

Figure 9 is a sequence listing of the coding sequence of AtMSH6, together with a deduced sequence of the encoded polypeptide.

Figure 10 is a protein alignment of yeast (Saccharomyces cerevisiae) and Arabidopsis thaliana MSH6 protein.

Figure 11 is a genomic sequence listing of AtMSH6.

Figure 12 is a plasmid map of plasmid pPFI3.

Figure 13 is a plasmid map of plasmid pPF14.

Figure 14 is a plasmid map of plasmid pCW186.

Figure 15 is a plasmid map of plasmid pCW187.

Figure 16 is a plasmid map of plasmid pPF66.

Figure 17 is a plasmid map of plasmid pPF57.

Figure 18 is a diagrammatic representation of an antisense gene construction for use in homeologous meiotic recombination.

Figure 19 is a plasmid map of plasmid p32 4 3.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the inventors' discovery that there exist in higher plants genes which are homologous to MMR genes in E. coli, and to MMR genes in yeasts and humans.

Thus, the inventors have identified genes, herein designated AtMSH3 and AtMSH6, of the plant Arabidopsis thaliana which encode the proteins AtMSH3 and AtMSH6.

These plant proteins are homologous to yMSH3 and yMSH6, respectively. The present inventors have isolated cDNAs encoding the proteins AtMSH3 and AtMSH6 and have isolated the complete gene encoding AtMSH6. Given the teaching herein, other genes (for example AtMSH2, and genes of other plants) may be obtained which are involved in DNA mismatch repair in plants, including other genes which encode polypeptides homologous to MMR proteins of yeasts or humans, such as genes which encode WO 99/19492 PCT/EP98/06977 8 polypeptides homologous to yeast MSH2. MLHI or PMS2. or to human MLHL. PMSI or PMS2. For example, given the teaching herein, genes of members of the Brassicaceae family or of other unrelated families, for example the Poaceae. the Solanaceae, the Asteraceae, the Malvaceae. the Fabaceae, the Linaceae. the Canabinaceae, the Dauaceae and the Cucurbitaceae family, and which encode polypeptides homologous to MMR proteins of yeasts or humans may be obtained.

Examples of plants whose genes encoding polypeptides homologous to MMR proteins of yeasts or humans may be obtained given the teaching herein include maize, wheat, oats, barley, rice, tomato, potato, tobacco, capsicum, sunflower, lettuce, o1 artichoke, safflower, cotton, okra, beans of many kinds including soybean, peas, melon, squash, cucumber, oilseed rape. broccoli, cauliflower, cabbage, flax, hemp, hops and carrot.

Within the meaning of the present invention, a first polypeptide is defined as homologous to a second polypeptide if the amino acid sequence of the first polypeptide exhibits a similarity of at least 50% on the polypeptide level to the amino acid sequence of the second polypeptide.

A procedure which may be followed to obtain genes AtMSH3 and AtMSH6 is described in Example 1. Essentially the same technique may be applied to obtain other mismatch repair genes of Arabidopsis thaliana, and essentially the same technique as exemplified herein may be applied to cDNA obtained by reverse transcription of RNA from other plants. Alternatively, given the sequence information disclosed herein, other degenerate oligonucleotide primers, especially oligonucleotides of the invention which are capable of hybridising at 45 0 C under standard PCR conditions (such as the conditions described in Example 1 using primers UPMU and DOMU) to AtMSH3 and/or AtMSH6 may be designed and obtained for use in isolating sequences of plant mismatch repair genes which are homologous to AtMSH3 or AtMSH6, from other plants. Similarly, oligonucleotides of the invention which are capable of hybridising at 45 0 C under standard PCR conditions to plant mismatch repair genes of plants other than Arabidopsis thaliana also fall within the scope of the present invention and may be utilised to obtain mismatch repair genes of still other plants. Typically, such oligonucleotides are capable of hybridising at 45 0 C under standard PCR conditions to a DNA molecule which encodes a polypeptide which is homologous to a mismatch repair polypeptide of a yeast or a human.

The temperature at which oligonucleotides of the invention hybridise to AtMSH3 and/or AtMSH6, or to plant mismatch repair genes of plants other than Arabidopsis thaliana, or to DNA molecules which encode polypeptides which are homologous to a mismatch repair polypeptide of a yeast or a human may be higher than 45 0 C, for example at least 50°C, or at least 55 0 C, or at least 60*C or as high as WOn 00/19492 PCT/EP98/06977 9 The successful gene isolation disclosed herein demonstrates for the first time the existence of MMR in higher plants and indicates the presence of other plant MMR genes.

For example, genes encoding the plant homologs of MSH1. MSH2. MSH4, PMSI. PMS2 and MLHI may be identified given the teaching herein. Such genes, as well as those specifically described herein, separately or in combination, are useful in manipulating the plant MMR for plant breeding purposes. Thus, for example, the plant MMR may be altered by including in a plant cell a polynucleotide sequence as defined herein-above with reference to the third embodiment of the invention, and causing the polynucleotide sequence to express either a polynucleotide which disables a plant MMR gene, or a polypeptide which disrupts the plant's MMR system.

The DNA molecule of the third embodiment of the invention includes a polynucleotide sequence (herein referred to as a MMR altering gene) which may for example encode sense, antisense or ribozyme molecules characterised by sufficient base sequence similarity or complementarity to the gene to be altered to permit the antisense or ribozyme molecule to hybridise with the plant MMR gene in vivo or to permit the sense molecule to participate in co-suppression. Alternatively, the MMR altering gene may encode a protein or proteins which interfere with the activity of a plant MMR protein and thus disrupt the plant's MMR system. For example, such encoded proteins may be antibodies or other proteins capable of interfering with MMR protein function, such as by complexing with a protein functionally involved in plant MMR thereby disrupting the MMR of the plant. An example of such a protein is the MSH3 protein of Arabidopsis thaliana described herein or a protein of another plant which is homologous to the MSH3 protein of A. thaliana. For instance, overexpression of MSH3 in a plant cell causes MSH2 present in the cell to be substantially completely complexed, disrupting the mismatch repair mechanism or mechanisms in the cell which are functionally dependent on the presence of a complex of MSH2 with MSH6. Similarly, mismatch repair mechanisms which depend on the presence of a complex of MSH2 and MSH3 may be disrupted by the overexpression of MSH6.

A chimeric gene of the fourth embodiment, incorporating a MMR altering gene, may be prepared by methods which are known in the art. Similarly, the MMR altering gene may be introduced into a plant cell, regenerating tissue or whole plant by techniques known in the art as being suitable for plant transformation, or by crossing. Known transformation techniques include Agrobacterium tumefaciens or A. rhizogenes mediated gene transfer, ballistic and chemical methods, and electroporation of protoplasts.

The MMR altering gene or genes are typically expressed from suitable promoters.

Suitable promoters may direct constitutive expression, such as the 35S or the NOS promoter. Usually, however, the promoter will direct either inducible or tissue specific callus; embryonic tissue; etc.), cell type specific protoplasts; meiocytes; etc.) or developmental embryo) expression of the altering gene or genes, in order for the W 9 00/104A9 PCT/EP98/06977 MMR system to function in tissue types or cell types, or at developmental stages of the plant. in which it is not desirable for the MMR system to be altered. Using such promoters, therefore, the activity of a MMR altering gene may be limited to a specific stage during plant development or it may be altered by controlling conditions external to the plant, and the deleterious effects of a permanently disabled or altered DNA mismatch repair system in a plant may be avoided. Examples of suitable promoters which are not constitutive are known in the art and include inducible promoters such as PRla (reviewed by Gatz, 1997, Annual Rev. Plant Phys. Plant Mol. Biol. 48: 89), tissue specific promoters such as AoPRI (Sabahattin et al., 1993, Biotechnology 11: 218), and cell-type specific promoters such as DMC1.

A chimeric gene in accordance with the invention may further be physically linked to one or more selection markers such as genes which confer phenotypic traits such as herbicide resistance, antibiotic resistance or disease resistance, or which confer some other recognisable trait such as male sterility, male fertility, grain size, colour, growth is rate. flowering time, ripening time. etc.

The process of the tenth embodiment of the invention provides, for example, a process for generating intraspecies genetic variation by altering the mismatch repair system in a plant cell, in regenerating plant tissue or in a whole plant. The plant cell, regenerating tissue or whole plant includes and expresses one or more MMR altering genes which are capable of altering mismatch repair in the plant cell, regenerating tissue or whole plant. Alteration of MMR may be achieved, for example, by inactivating the genes encoding plant MSH3 and/or plant MSH6. It is preferred to inactivate the plant MSH3 and MSH6 encoding genes at the same time and in the same plant cell, regenerating tissue or whole plant. Typically in this preferred form of the invention inactivation of either plant MSH3 or MSH6 alone is insufficient to substantially alter the plant's mismatch repair system and only when both MSH3 and MSH6 are inactivated simultaneously is the plant's mismatch repair system sufficiently altered to prevent the MMR system from recognising base pair mismatches, base insertions or deletions as a result of DNA replication errors, DNA damage, or oligonucleotide induced site-specific mutagenesis. However, in some applications of the invention, inactivation of only one gene may also be used to cause genomic instability or increase the efficiency of sitespecific mutagenesis.

If desired, the MMR altering gene or genes may later be rendered non-functional or ineffective, or may be removed from the genome of the plant cell, regenerating tissue or whole plant in order to restore mismatch repair in the plant cell, regenerating tissue or whole plant. The MMR altering gene or genes may be inactivated by means of known gene inactivation tools, such as ribozymes, or may be removed from the genome using gene elimination systems known in the art, such as CRE/LOX. It is preferred to render two genes. whose gene products combine to incapacitate MMR, ineffective by separating ^hf r J AJ* PCT/IP /07;O7 WU "YiY4Y 11 ,I w the altering genes through segregation. Therefore, in a preferred embodiment of the invention a first plant cell or plant is generated in which only plant MSH3 is incapacitated.

and a second plant cell or plant is generated in which only plant MSH6 is incapacitated.

The combination of both genomes. for example by crossing, then produces significant s MMR deficiency in those cells or plants which have inherited both altering genes. If the altering genes are expressed from unlinked loci, gene segregation restores MMR activity in the progeny of the cells or plants.

In a process of the ninth embodiment of this invention, homeologous recombination is enhanced between different genomes, chromosomes or genes in plant cells or plants by o1 altering MMR in said plant cells or plants. Such genomes, chromosomes or genes are characterised in that they originate from different plant families, genera, species, subspecies, plant varieties or lines. Hybrid plant cells or hybrid plants may be produced by crossing, by cell fusion or by other techniques known in the art. These plant cells or plants are further characterised by expressing one or more genes that are capable of altering mismatch repair in the plant cell or plants.

In the process of the ninth embodiment. the homeologous recombination is typically for the purpose of introducing a desired characteristic in the hybrid plant. In this typical application of the process of the ninth embodiment, and in the process of the tenth embodiment the desired characteristic may be any characteristic which is of value to the plant breeder. Examples of such characteristics are well known in the art and include altered composition or quality of leaf or seed derived storage products oil, starch, protein), altered composition or quality of cell walls decrease in lignin content), altered growth rate, prolonged flowering, increased plant yield or grain yield, altered plant morphology, resistence to pathogens, tolerance to or improved performance under environmental stresses of various kinds, etc.

In a preferred form of the tenth embodiment, an MMR altering gene is cointroduced along with the homeologous genome, chromosome or gene of another plant cell or plant into an MMR proficient plant cell or MMR proficient plant to produce a hybrid plant cell or hybrid plant in which homeologous recombination can occur.

Suitably, the MMR proficient plant cell or MMR proficient plant may also include an MMR altering gene. For example a gene capable of inactivating plant MSH3 may be cointroduced along with the homeologous genome, chromosome or gene of another plant cell or plant into an MMR proficient plant cell or MMR proficient plant in which MSH6 is inactivated. A resultant hybrid plant in which homeologous recombination occurs will as include both the MSH3 and MSH6 altering genes and its MMR system will therefore be inactivated.

In this form of the invention, if hybrid plants are to be produced by crossing, the MMR altering gene preferably originates from the male parent, thus ensuring that the ixif-t nnf'n n'^ PrCT/iVD 1I/ 177 WU YYiy IYL- 12 MMR altering gene is always introduced and is not present in the recipient cell. That is, the MMR of the recipient cell. prior to introduction of the MMR altering gene. is typically proficient. Alternatively, if an MMR altering gene is present in a recipient cell it may be ineffective or inefficient on its own. or it may be linked to an inducible or tissue s specific or cell type specific promoter which only renders the MMR altering gene active under limited conditions.

Thus, in a preferred form of the process of the ninth embodiment, the MMR system of the hybrid plant is initially unaltered. In this form of the process, the step of altering the mismatch repair system may comprise introducing into the hybrid plant, or cells thereof, a MMR altering gene, such as by Agrobacterium tumefaciens or A. rhizogenes mediated gene transfer, ballistic and chemical methods, and electroporation of protoplasts.

The MMR altering gene or genes are typically expressed from suitable promoters, as described above. Preferably, the promoter is transcriptionally active in mitotically and meiotically active tissue and/or cells to ensure MMR alteration after chromosome pairing at mitosis and meiosis, respectively. The preferred timing for MMR alteration is at meiosis, because recombinant genomes. chromosomes or genes are directly transmitted to the progeny. A suitable meiocyte specific promoter is for example the DMC1 promoter from Arabidopsis thaliana ssp. Ler. (Klimyuk and Jones, 1997, Plant J. 11, 1-14).

However, mitotic homeologous recombination is also a desirable outcome as somatic recombination events can be transmitted to offspring due to the totipotency of plant cells and the lack of predetermined germ cells in plants.

If desired, the MMR altering gene or genes may later be rendered non-functional or ineffective, or may be removed from the hybrid plant or hybrid plant cells, in order to restore mismatch repair in the hybrid plant or hybrid plant cells. The MMR altering gene or genes may be inactivated by means of known gene inactivation tools as described herein above.

EXAMPLES

Example 1. Cloning of the AtMSH3 and AtMSH6 coding sequences Isolation of partial AtMSH3 and AtMSH6 consensus sequences Degenerate oligonucleotides UPMU (SEQ ID NO:I) and DOMU (SEQ ID NO:2) UPMU CTGGATCCACIGGICCIAA(C/T)ATG DOMU CTGGATCC(A/G)TA(A/G)TGIGTI(A/G)C(A/G)AA were used to isolate AtMSH3 and AtMSH6 sequences by PCR amplification.

Primers UPMU and DOMU correspond to conserved amino acid sequences of the proteins MutS coli and S. typhimurium), HexA pneumoniae). Repl (mouse) and Duc l (human). The conserved regions to which they are targeted are TGPNM for UPMU (amino acid positions 852-856 for AtMSH6 and 816-820 for AtMSH3) FATHY or FVTHY wn O9/i a00 Pr'T/IPo/no77 13 for DOMU (amino acid positions 964-968 for AtMSH6 and 928-932 for AtMSH3.

respectively.) These primers have been used to isolate MSH2 and MSHI from yeast (Reenan and Kolodner, Genetics 132: 963-973 (1992)) and MSH2 from Xenopus and mouse (Varlet et al.. Nuc. Acids Res. 22:5723-5728 (1994)).

Template single strand cDNA was produced by reverse transcription of 2 utg total RNA from a cell suspension culture of Arabidopsis thaliana ecotype Columbia (Axelos et al. 1989, Mol. Gen. Genetics 219: 106-112). The PCR reaction was performed under the following conditions in a final volume of l00pl: 0.2mM dNTP, 1pM each primer, IXPCR buffer, lu Taq DNA polymerase (Appligene) in the presence of template cDNA. PCR parameters were 5 minutes at 94°C, followed by 30 cycles of 40 seconds at 95 0 C, seconds at 45 0 C, 1 minute at 72 0 C. The amplification products were cloned into pGEM-T vector (Promega) and sequenced. Two different clones were isolated, S5 (350bp) was homologous to MSH3, S8 (327bp) was homologous to MSH6. Complete cDNA sequences were then isolated according to the Marathon cDNA amplification kit procedure (Clontech).

In summary. this procedure involves producing double stranded cDNA by reverse transcription of 2Lg polyA+ RNA from the cell suspension culture of Arabidopsis.

Adaptors are ligated on each side of the cDNA. The ligated cDNA is used as a template for and 3' RACE PCR reactions in the presence of primers that are specific for the adaptor on one side (API and AP2), and specific for the targeted gene on the other side. A 5' and a 3' fragment that overlap are thus produced for each gene. The complete gene coding sequence can be reconstituted taking advantage of a unique restriction site, if available, in the overlapping region. Specific details of this procedure as it was used to isolate AtMSH3 and AtMSH6 coding regions, are as follows.

Isolation of AtMSH3 complete coding sequence From the sequence of clone S5, primer 636 (SEQ ID NO:3) was designed: 636 TGCTAGTGCCTCTTGCAAGCTCAT.

Primer API (SEQ ID NO:4) is complementary to a portion of an adaptor sequence which had been ligated to the 5' and 3' ends of Arabidopsis cDNA: API CCATCCTAATACGACTCACTATAGGGC.

PCR performed on the ligated cDNA with primers 636 and API for the 5' RACE PCR was followed by a second round of amplification with the nested primers AP2 (SEQ ID and S525 (SEQ ID NO:6) AP2 ACTCACTATAGGGCTCGAGCGGC S525 AGGTTCTGATTATGTGTGACGCTTTACTTA (the latter was also designed to correspond to a part of the sequence of clone S5) and produced a 2720bp DNA fragment. Figure 1 provides a diagrammatic representation of the primer sequences used to isolate AtMSH3. Another primer (S51, SEQ ID NO:7) S51 GGATCGGGTACTGGGTTTTGAGTGTGAGG WO 99/19492 PCT/EP98/06977 14 was designed closer to the 5' border and permitted the determination of 99bp upstream to the AT(; initiation codon. For the 3' RACE PCR. a first PCR reaction was performed with primers API and 635 (SEQ ID NO:8).

635 GCACGTGCTTGATGGTGTTTTCAC followed by a second round of amplification, using the nested primers AP2 and S523 (SEQ ID NO:9) S523 TCAGACAGTATCCAGCATGGCAGAAGTA which produced a DNA fragment of 890bp. Both DNA fragments were subcloned into pGEM-T and sequenced. Since PCR amplification using the Expand Long Template PCR o1 System (Boehringer-Mannheim) produced errors in the sequence, new oligonucleotides were designed to isolate those sequences again by PCR, but with the high fidelity DNA polymerase Pfu. PCR with primers 1S5 (SEQ ID NO:10) and S53 (SEQ ID NO:11)

ATCCCGGGATGGGCAAGCAAAAGCAGCAGACGA

S53 GACAAAGAGCGAAATGAGGCCCCTTGG amplified the 1244bp fragment clone 52 (SEQ ID NO:12. cloned into pUC18/Smal).

PCR

with primers S52 (SEQ ID NO:13) and 2S5 (SEQ ID NO:14)

ATCCCGGGTCAAAATGAACAAGTTGGTTTTAGTC

S52 GCCACATCTGACTGTTCAAGCCCTCGC amplified the 2104bp clone 13 (SEQ ID NO:15, cloned into pUC18/Smal). The complete coding sequence of the AtMSH3 gene was reconstructed in pUC18 by ligating the 5' half of AtMSH3 (clone 52) to the 3' half of AtMSH3 (clone 13) after digesting with BamH1 which has a unique cleavage site in the overlapping region of both clones. This manipulation yielded plasmid pPF26. The Smal fragment from pPF26 contains the complete AtMSH3 coding sequence. The remaining primers referred to in Figure 1 are as follows: S51 GGATCGGGTACTGGGTTTTGAGTGTGAGG (SEQ ID NO:16) S525 AGGTTCTGATTATGTGTGACGCTTTACTTA (SEQ ID NO: 17) Figures 2 and 3 provide plasmid maps of clones 52 and 13 respectively, showing restriction enzyme cleavage sites. The complete AtMSH3 coding sequence (SEQ ID NO:18) is 3246bp long and is shown in Figure 4 together with the deduced sequence (SEQ ID NO:19) of the encoded polypeptide. AtMSH3 is clearly homologous to the yeast and mouse MSH3 genes. A sequence alignment of polypeptides encoded by AtMSH3 and that encoded by Saccharomyces cerevisiae MSH3 is set out in Figure Isolation of the AtMSH6 complete coding sequence and genomic sequences The same procedure allowed isolation of the AtMSH6 cDNA. Figure 6 provides a diagrammatic representation of the primer sequences used to isolate AtMSH6. For the RACE PCR, primers 638 (SEQ ID NO:20) and API (SEQ ID NO:4) 638 TCTCTACCAGGTGACGAAAAACCG allowed the amplification of a 2889 DNA fragment. Primer S81 (SEQ ID NO:21) 11-\ 1QIAAO) PCT/FPOR/f6977 S81 CGTCGCCTTTAGCATCCCCTTCCTTCAC helped define the 142bp upstream to the ATG initiation codon. On the 3' side. RACE PCR was initially performed with primers S823 (SEQ ID NO:22) and API (SEQ ID NO:4), S823 GCTTGGCGCATCTAATAGAATCATGACAGG s and then with the nested primers 637 (SEQ ID NO:23) and AP2 (SEQ ID 637 GACAGCGTCAGTTCTTCAGAATGC to produce a 774bp DNA fragment. As for AtMSH3, those fragments were cloned and sequenced. Re-isolation of the DNA sequence using the high fidelity Pfu polymerase and newly designed primers 1S8 (SEQ ID NO:24) and S83 (SEQ ID NO:25) (for the 5' side) led to a 2182 bp DNA fragment identified as clone 43 (SEQ ID NO:26, cloned in pUC18/Smal), and a 1379bp clone identified as clone 62 (SEQ ID NO:27, also cloned in pUC18/Smal).

1S8 ATCCCGGGATGCAGCGCCAGAGATCGATTTTGT 2S8 ATCCCGGGTTATTTGGGAACACAGTAAGAGGATT (SEQ ID NO:28) S82 GCGTTCGATCATCAGCCTCTGTGTTGC (SEQ ID NO:29) S83 CGCTATCTATGGCTGCTTCGAATTGAG Figures 7 and 8 provide plasmid maps of clones 43 and 62 respectively, showing restriction enzyme cleavage sites. Clones 43 and 62 were digested by the XmnI restriction enzyme for which a unique site is present in their overlapping region and then ligated. The complete AtMSH6 coding sequence (SEQ ID NO:30) is 3330bp long and is shown in Figure 9 together with the deduced sequence (SEQ ID NO:31) of the encoded polypeptide. AtMSH6 is clearly homologous to the yeast and mouse MSH6genes. A sequence alignment of polypeptides encoded by AtMSH6 and that encoded by Saccharomyces cerevisiae MSH6 is set out in Figure An AtMSH6 genomic sequence was also isolated from a genomic DNA library constituted after partial Sau3AI digestion of DNA from the Arabidopsis cell suspension.

8062bp were sequenced that covered the AtMSH6 gene and show colinearity with the cDNA. 16 introns are found scattered along the gene. The complete genomic sequence (SEQ ID NO:98) is shown in Figure 11.

Example 2. A measure of somatic variation in MMR deficient plants Constructs Constructs with antisense AtMSH3 or antisense AtMSH6 or both AtMSH3/AtMSH6 under the control of a single 35S promoter have been inserted into the binary vector pPZP121 (Hajdukiewicz et al., Plant Mol. Biol. 23, 793-799) between the right and left borders of the T-DNA. The pPZP121 plasmid confers chloramphenicol resistance to Escherichia coli or Agrobacterium tumefaciens bacteria. The aacCl gene is carried by the T-DNA and allows selection of transformed plant cells on gentamycin (Hajdukiewicz et al., Plant Mol. Biol. 25, 989-994). For the purpose of expressing antisense constructs. a i/-r fr I~j^nrk PC"T/IP9DO/1077 WU yy/ IIYQL 16 .0 promoter/terminator cassette with a polylinker was introduced into pPZPl2l. The 3' ends of the considered genes have been chosen since this region seems more efficient for antisense inhibition. For AtMSH3 this corresponds to clone 13 (2104bp). for AtMSH6 this corresponds to clone 62 (1379bp). Clone 13 comprises 2104bp of the 3' region that were cut off the pUCl8 vector by Sall/Sstl restriction, blunted with T4 DNA polymerase and ligated into the T4 DNA polymerase blunted BamHI site of pPZP121/35S, creating clone pPF13.

The same procedure was followed for the 3' region of AtMSH6 clone 62 (1379bp) thus creating plasmid pPF14. For the double constructs, the 3' region (from clone 62) of AtMSH6 was introduced ahead of the AtMSH3 region into pPF13 creating pCW186 and to reciprocally, the 3' region of AtMSH3 (from clone 13) was introduced ahead of AtMSH6 into pPF 14, creating pCW187.

These constructs were introduced into the Arabidopsis cells (as described below) of wildtype Columbia and of the Columbia tester line.

An alternative strategy to antisense inhibition of AtMSH6 comes from experiments of Marra et al. (1998. Proc. Natl. Acad. Sci USA 95. 8568-8573) who show that overexpression of functional MSH3 results in depletion of MSH6 protein in human cells.

This depletion may generate a mismatch repair mutant phenotype.

For the purpose of overexpressing functional AtMSH3 protein in plant cells, the complete MSH3 coding region was excised from pPF26 (example 1) by digestion with Smal, and was inserted into the SmaI site of pCW164. The resulting construct was named pPF66. It contains a complete AtMSH3 gene under the control of the 35S promoter inside the left (LB) and right (RB) border of the T-DNA. This T-DNA also contains the hpt2 gene for gentamycin selection. Plasmid pPF66 was introduced into Arabidopsis cells as described below. One cell clone was selected which clearly overexpressed the AtMSH3 gene as shown by Northern analysis. Figures 12-16 provide plasmid maps of plasmids pPF13, pPF14, pCW186, pCW187 and pPF66, respectively.

Construction of tester construct For the purpose of Forward Mutagenesis Assays, a tester construct was built containing the coding regions for nptlI, codA, uidA. All three genes are driven by the promoter and are terminated by the 35S terminator. This construct was obtained by introducing an EcoRl fragment encoding the codA cassette (2.5kb) and a HindIII fragment encoding the uidA (GUS) cassette (2.4kb) into the pPZPIll vector (Hajdukiewicz et al.,1994, Plant Mol Biol 23: 793-799) which already contained the nptlI expression cassette.

This new plasmid was named pPF57. NptII is used to select for transformed plant cells.

GUS is used to analyse the degree of gene silencing in the construct to identify cell lines in which the transgenes are expressed), and codA is used as a marker for forward mutagenesis (described below).

Wn 00/1 QA4 PCT/EP98/06977 17 The plasmid map of pPF57 is provided in Figure 17.

Plant cell transformation The constructs are introduced into Agrohucterium by electroporation. Plant cells are then transformed by co-cultivation. A suspension culture of Arabidopsis thaliana cells that has been established by Axelos et al. (1992, Plant Physiol. Biochem. 30, 1-6) may be used.

One day old freshly subcultured cells are diluted five times into AT medium (Gamborg medium. 30g/l sucrose, 200.g/l NAA). 10ll of saturated Agrobacterium containing the transforming T-DNA constructs are added to 10ml diluted cells in a 100ml erlenmeyer. The co-cultivation is agitated slowly (80rpm) for 2 days. The cells are then washed 3 times into AT medium and finally resuspended in the same initial volume (10ml). The culture is agitated for 3 days to allow expression before plating on selection plates (AT/BactoAgar 8g/l+gentamycin 50pg/ml). Transformed individual calli are isolated 3 weeks later.

Tester Strain The tester construct on plasmid pPF57 was introduced into Arabidopsis cells of 1 wildtype Columbia using the transformation protocol described above. Among 10 candidate transformants. one cell clone was shown (by Southern analysis) to have a unique T-DNA insertion. All three genes were shown to be functional in this cell line as indicated by resistance to kanamycin, blue staining in the presence of X-Glu (GUS), and sensitivity to (codA).

MMR altering genes (described above) were then introduced individually into the tester line and transformed cells are used for analysis of both Microsatellite Instability and Forward Mutagenesis.

Microsatellite analysis Microsatellites have been described in Arabidopsis (Bell and Ecker, 1994, Genomics 19. 137-144). The present Example is based on a study of instability of microsatellites that are polymorphic amongst different ecotypes. DNA is extracted from the transformed calli.

Specific primers have been defined that are used to amplify the microsatellite sequence.

One of the two primers is previously p 32 labelled by T4 kinase. In case of a polymorphic variation, new PCR products appear that do not follow the expected pattern of migration on a polyacrylamide gel. This is a commonly observed feature for MMR deficient cells in yeast or mammalian cells.

In particular, the present Example describes a study on microsatellites ca72 (CA 1 8 ngal72 (GA 29 and ATHGENEA(A 39 chosen because they belong to the types predominantly affected in human mismatch repair deficient tumors. The size of these microsatellites is not conserved from one Arabidopsis ecotype to the other.

Arabidopsis cells which are transformed with an MMR altering gene (above) and control cells not expressing the MMR altering gene are allowed to form calli. DNA is WO 99/19492 18 PCT/EP98/06977 rapidly extracted from the calli and is analysed for microsatellite instability as described in detail by Bell and Ecker 1994. Genomics 19. 137-144. In summary, the relevant microsatellite is amplified by PCR using P32 labelled primers. The PCR products are separated on a DNA sequencing gel for size determination. Size differences between microsatellites from transformed and control cells not expressing the MMR altering gene in question indicate microsatellite instability as a result of MMR alteration.

The sequences of primers used for PCR amplification of microsatellites ca72 and ngal72 are included in Table 1. PCR amplification of microsatellite ATHGENEA made use of a forward primer containing the sequence to ACCATGCATAGCTTAAACTTCTTG (SEQ ID NO:32) and of a reverse primer containing the sequence ACATAACCACAAATAGGGGTGC (SEQ ID NO:33).

The amplification for microsatellite ca72 revealed in Columbia control cells (with respect to the MMR altering gene) a 248 bp long PCR fragment instead of the published is length of 124 bp. DNA sequencing verified this fragment as a CAm microsatellite.

Forward mutagenesis assay Tester cells transformed with antisense .AtMSH3 or antisense AtMSH6 or both AtMSH3/AtMSH6 are analysed for the stability of the codA gene. The functional codA gene confers to sensitivity to 5-fluoro-cytosine (5FC). whereas a gene inactivating mutation in codA will confer resistance to 5FC. The frequency of resistant cells is therefore a good indicator of somatic variation as a direct result of MMR alteration. Variants resistant to are first analysed for GUS activity. IfGUS is inactive. 5FC resistance is assumed to be due to gene silencing (all three genes are under the 35S promoter). If GUS is active, resistance is assumed to be due to forward mutations that have inactivated codA. PCR is then performed on the putative codA mutant genes which is then sequenced to confirm the presence of forward mutations in codA.

Besides codA, other marker genes may also be used for the Forward Mutagenesis Assay such as the ALS gene (conferring sensitivity to valine or to sulfonylurea; Hervieu and Vaucheret, 1996, Mol. Gen. Genet. 251 220-224: Mazur et al. 1987, Plant Physiol. 85 1110- 1117).

Example 3. Homeologous meiotic recombination in Arabidopsis thaliana A. Construction of a meiocvte specific gene expression cassette comprising the DMC1 promoter and the NOS terminator The DMC1 promoter may be used as published by Klimyuk and Jones, 1997, Plant J. 11.1-14). To obtain a more convenient alternative for gene cloning, a 3.3 Kb WO 00/1 qA PCT/EP98/06977 19 long subfragment of the DMCI promoter was obtained by PCR from genomic DNA of Arabidopsis thaliana (ssp. Landsberg erecta "Ler").

The PCR was done in three rounds: Round One: A 3.7 Kb long product was obtained using the forward primer DMCIN-A comprising the sequence GAAGCGATATTGTTCGTG (SEQ ID NO:34) and the reverse primer DMCIN-B comprising the sequence AGATTGCGAGAACATTCC (SEQ ID The weak amplification product was then used as template for round two and three.

Round Two: A 3.1 Kb long product comprising the promoter and the untranslated leader was obtained using forward primer DMCIN-1. which contained the sequence acgcgtcgacTCAGCTATGAGATTACTCGTG (SEQ ID NO:36) and introduced a Sall cloning site at the 5' end of the promoter fragment, and reverse primer DMCIN-2 which contained the sequence gctctagaTTTCTCGCTCTAAGACTCTCT (SEQ ID NO:37) and introduced a XbaI site at the 3' end of the PCR fragment.

Round Three: A 0.2 Kb long product comprising the first exon/intron of the DMC1 promoter was obtained using forward primer DMCIN-3, which contained the sequence gctctagaGCTTCTCTTAAGTAAGTGATTGAT (SEQ ID NO:38) and introduced a XbaI site at the 5' end of the PCR fragment, and reverse primer DMCIN-4, containing the sequence tcccccgggctcgagagatctccatggTTTCTTCAGCTCTATGAATCC (SEQ ID NO:39) and introduced at the 3' end of the PCR product restriction sites for NcoI. Bglll. XhoI and SmaI.

The products obtained in round Two and Three were digested with Xbai and subsequently ligated to reconstitute a 3.3 Kb long DMC1 promoter from which the first two in-frame ATG start codons were replaced with a unique restriction site for XbaI.

This promoter can be cloned between the restriction sites for Sall and Smal of p 3 2 6 4 which contains the SacI-EcoRI NOS terminator in pBIN19, to yield the entire expression cassette in pBIN19. This cassette contains the following cloning sites: Ncol, BgllI, XhoI.

SmaI and (already present on p3264) KpnI and SacI.

(ii) Another strategy yielded the following convenient DMC1 promoter. A 1.8 kb DNA fragment comprising the 3' terminal part of the meiocyte specific DMC1 promoter was isolated by PCR from purified genomic DNA of Arabidopsis thaliana (ssp. Landsberg erecta The forward PCR primer (DMCla) contained the sequence acgcgtcgacGAATTCGCAAGTGGGG (SEQ ID and introduced a Sall cloning site at the 5' end of the promoter fragment. The reverse PCR primer (DMClb) contained the sequence lO/ 9 QQ41AQ PCT/I~Po/flM77 tccatggagatctcccgggtacCGATTTGCTTCGAGGG (SEQ ID NO:41) introducing a polylinker region at the 3' end of the promoter fragment. The PCR reaction was carried out with VENT DNA Polymerase (NEB) over 25 cycles using the following cycling protocol: 1 minute at 94 0 C, 1 minute at 54 0 C, 2 minutes at 72 0

C.

The PCR reaction yielded a blunt ended DNA fragment which was digested with restriction enzyme Sall and was cloned into the cleavage sites of restriction enzymes Sail and Smal in plasmid p2030, a pUC118 derivative containing the SacI-EcoRI NOS terminator fragment of pBIN121. The cloning yielded plasmid p2031, containing the DMCI promoter-polylinker-NOS terminator expression cassette depicted in Figure 18.

o1 B. Construction of an MSH3 antisense gene under the control of the DMC1 promoter A 2.1 kb DNA fragment encoding the carboxyterminal part of AtMSH3 was removed from the polylinker of clone 13 described in Example 1 after digestion with KpnI. (ii) blunting of the DNA ends generated by KpnI and (iii) digestion with BamHI.

The isolated fragment was then cloned in antisense orientation downstream of the DMC1 promoter in plasmid p 2 0 3 1 which had been digested with restriction enzymes SmaI and BgllI. This cloning yielded plasmid p2033 (Figure 18).

After digestion of p2033 with EcoRI, a 4.1 kb DNA fragment was recovered comprising the DMC1 promoter, the partial MSH3 cDNA in antisense orientation with respect to the promoter and the NOS terminator. This fragment was cloned into the EcoRI restriction site of plant transformation vector pNOS-Hyg-SCV to yield plasmid p3242 (Figure 18).

C. Construction of a combined MSH6/MSH3 antisense gene under the control of a single DMCI promoter A 3.1 kb fragment, encoding in antisense orientation the partial AtMSH6 and AtMSH3 sequences and the 35S terminator, was isolated from pCW186 by digestion with KpnI.

The fragment was treated with Klenow enzyme to blunt both ends. It was then cloned into the SmaI site of plasmid p3243 (a pNOS-Hyg-SCV derivative, illustrated in Figure 19), which had been opened to delete the region between the SmaI sites. Clones containing the fragment in the antisense orientation with respect to the DMC1 promoter (described in A(ii) above) were identified by diagnostic digestion with BamHI. The selected construct was named p3261.

Another practical way of cloning the double antisense gene is as follows. A 1 kb DNA fragment encoding the carboxyterminal part of AtMSH6 is isolated from clone 62 described in Example 1 after digestion of clone 62 plasmid DNA with BamHI, which cleaves in the 5' polylinker region flanking the partial cDNA, and with EcoRI, which cleaves within the cDNA. The isolated fragment is treated with Klenow enzyme to blunt both its ends and is cloned into the recipient plasmid p2033 or p3242. For the purpose of \If1 QQ9n nAQ) PrTE/V DO nO'77 Jd 7n.J711I7*7 21 *A-1i 701U 721 cloning, the recipient plasmid may be cleaved with either Aval or Ncol and can be blunted with Klenow enzyme to produce blunt acceptor ends for fragment cloning. This cloning yields two opposite orientations of cloned fragment DNA with respect to the DMC1 promoter. These can be identified by diagnostic digestion with restriction enzymes ScaI or XmnI in conjunction with Sacl. The selected construct contains the DMC1 promoter, the combined partial cDNAs for AtMSH3 and AtMSH6 (both cloned in antisense orientation with respect to the DMC1 promoter) and the NOS terminator. If the recipient plasmid is p2033, the combined antisense gene under control the single DMC1 promoter is recovered from the vector after EcoRI digestion and is cloned into the EcoRI restriction site of pNOS-Hyg-SCV.

D. Construction of a full-length MSH3 sense gene under control of the DMC1 promoter for overexpression of functional MSH3 protein Overexpression of MSH3 protein was shown in human cells (Marra et al., 1998, Proc. Natl. Acad. Sci. USA 95. 8568-8573) to complex all available MSH2 protein. This leaves MSH6 protein without its partner, leading to the degradation of MSH6 protein and eventually to a mismatch repair phenotype.

This phenomenon is exploited to increase homeologous meiotic recombination in Arabidopsis as an alternative to antisense inhibition of AtMSH6. For this purpose the fulllength cDNA encoding AtMSH3 is isolated from plasmid pPF66 by digestion with SmaI and is cloned into the SmaI site of the DMC1 expression cassettes described in A(i).

E. Selection of Recombination markers on homeologous chromosomes of Arabidopsis thaliana subspecies Landsbere erecta (Ler), Columbia (Col) and C24, respectively Visual recombination markers in Arabidopsis th. subspecies C24: Agrobacterium mediated transformation with a T-DNA containing a 35S-GUS gene, inactivated by insertion of a 35S-Ac transposable element (Finnegan et al., 1993, Plant Mol. Biol. 22, 625-633), had yielded a C24 line in which the T-DNA construct was integrated into chromosome 2. Genetic and molecular analysis of this line shows that the Ac transposon had excised from its T-DNA locus thereby restoring GUS activity, but had re-inserted into the chromosome at a distance of 16.4 cM, where it stayed fixed (due to disablement of Ac) within the chlorina gene. Insertional inactivation of the chlorina gene caused a bleached phenotype in those plants that were homozygous for this mutation.

Because of the two linked phenotypic markers, chlorina3A:Ac and GUS, this C24 line was used in crosses to wildtype Ler for analysis of meiotic homeologous recombination on chromosome 2 in conjunction with molecular recombination markers.

E(ii). Visual recombination markers in Arabidopsis th. Ler: The Ler line NW1 (obtained from NASC, Nottingham, UK) contains one recessive visual marker per chromosome, i.e. an-1 on Chr.1, py-1 on Chr.2, gll-1 on Chr.3, cer2-1 \W\l QQ/1Q40 PCT/~P98/06977 22 on Chr.4. and msl-I on Chr.5. This line is used in crosses to wildtype C24 which expresses an MMR altering gene for analysis of meiotic homeologous recombination on chromosomes 1-5 in conjunction with molecular recombination markers listed in Table 1.

Other Ler lines from NASC have several visual markers in close proximity to each other on the same chromosome. When these lines are used for hybrid production, analysis of homeologous meiotic recombination may make use entirely of visual recombination markers. Particularly suitable for crossing to C24 wildtype that is expressing a MMR altering gene are the following Ler lines: NW22: relative markers are disi (4 cM) ga4 (11 cM) thl on chromosome 1.

NW10: relevant markers are tz-201 (5 cM) cer3 on chromosome NW134, relevant markers are ttg (4 cM) ga3 on chromosome NW24 (abi3-1) and NW64 (gll-l). When present in the same plant on chromosome 3, abi3-1 and gll-i are 13 cM apart. Since this marker combination is not available from NASC. we have combined these markers by crossing line NW24 to line NW64. The Fl offspring were allowed to self-fertilise and to produce F2 seeds. F2 Plants which carry both markers as homozygous traits on the same chromosome can be identified firstly, by germinating F2 seeds on germination medium containing selective concentrations of abscisic acid, and subsequently, by identifying among the abscisic acid resistant plants those individuals which show the glabra phenotype.

E(iii) Molecular recombination markers in Col, Ler and C24: The genome of Arabidopsis thaliana is interspersed with unique base sequences arranged as simple tandem repeats. Allelic repeats can vary in length between different Arabidopsis subspecies and when amplified by PCR yield diagnostic DNA products of different length named Simple Sequence Length Polymorphisms (SSLPs). Many SSLPs have been genetically mapped and have been assigned to unique chromosome locations on the recombinant inbred map (Bell and Ecker. 1994, Genomics 19, 137-144; Lister and Deans lines, Weeds World 4i, May 1997).

In Table 1 are listed 28 mapped and established SSLPs between Ler and Col. A number of PCR primer pairs are described herein (SEQ ID NO:42 to SEQ ID NO:97) which also yielded SSLPs between C24 and Ler (19 SSLPs) or between C24 and Col SSLPs), respectively. Polymorphic SSLPs can be used as molecular markers in the analysis of homeologous recombination between genomes from these subspecies.

The PCR reactions (25 pL) were carried out over 35 cycles (15 seconds at 94°C, seconds at 55°C and 30 seconds at 72 0 with 0.25 U Taq DNA polymerase and 0.6 /tg genomic DNA in reaction buffer containing 2 mM MgCl2. PCR products were separated by agarose gel electrophoresis ultra high resolution agarose) and visualised by ethidiumbromide staining. The results from the PCR experiments are summarised in iL;/t 9o4iOA 9 PCT/EPR/n977 23 Table 1, which also shows the sequence of PCR primers, primer annealing temperature PCR product length and chromosome location of SSLP (with respect to the RI map of May 1997, Weeds World 4i).

F. Production of hybrid plants s C24 plants heterozygous for chlorina3A:Ac/GUS are crossed as male to emasculated wildtype Ler to produce Ler/C24(chlorina3A, GUS) hybrid seeds.

Due to the heterozygosity of the C24 parent, only 50 of hybrid plants have inherited the chlorina3A:Ac/GUS locus. The remaining 50% of hybrid plants are wildtype with respect to chlorina3A.Ac/GUS. Since the mutant locus is linked to a kanamycin to resistance gene (contained on the same T-DNA as GUS) mutant plants can be pre-selected by germinating hybrid seeds on germination medium containing 50 mg/L kanamycin.

Ler plants homozygous for the five chromosome markers are male sterile (msl-1) and are crossed without emasculation to wildtype C24 to produce Ler(an-1, py-1, gll-1, cer2-1, msl-l)/C24 hybrid seeds.

Other Ler plants. which are male fertile. are crossed after emasculation of the female parent to produce LerC24 hybrid seeds.

G. Introduction of MSH3 and MSH6/3 antisense genes into Arabidopsis and analysis of meiotic homeologous recombination Transformation of hybrid plants and analysis of homeologous meiotic recombination The plant transformation vectors comprising the antisense genes described in and above are introduced into Agrobacterium rumefaciens strain AGL1 (Lazo et al.. 1991, Bio/Technology 9, 963-967) by electroporation. Recombinant Agrobacterium clones are selected on LB medium containing 50 mg/L rifampicin and 100 mg/L carbenicillin.

Selected clones are used to infect roots of Arabidopsis hybrid plants (described in (F) above) using the root transformation protocol of Valvekens et al. (1988, PNAS 85. 5536- 5540) except that shoot and root inducing media contain hygromycin (10 mg/L) instead of kanamycin.

Plants regenerated from roots of hybrid plants are genetic clones of root donating plants and therefore are again genetic hybrids of two Arabidopsis subspecies described in However, in contrast to the root donating plants, the regenerated hybrid plants also contain the introduced transgene and the co-introduced hygromycin resistance gene with the latter allowing these plants to grow on hygromycin containing culture medium.

Hygromycin resistant plants are then allowed to enter the reproductive phase and to produce gametes by meiotic divisions of microspore and megaspore mothercells. At meiosis, the DMC1 promoter is activated and can direct the expression of antisense genes described in and above, leading to decreased MSH6 and/or MSH3 gene W' 10 A9 PCT/EP98/06977 S..24 expression. This in turn depletes the gamete mothercells of MSH6 and/or MSH3 protein, thus causing alteration of MMR during meiotic divisions and increasing the recombination frequency between homeologous chromosomes.

Transgenic plants are then allowed to self-fertilise and to produce seeds. These seeds (F2 seeds with respect to hybrid production), and the plants derived therefrom, carry the homeologous recombination events which can be identified by using the visual and molecular recombination markers described in above.

In case of homeologous recombination between chromosomes of Ler and C24(chlorina3A:Ac. GUS), the analysis concentrates on chromosome 2 by selecting plants showing the visual phenotypic marker chlorina. This marker thus serves as a reference point as it indicates that respective chromosomes 2 originate from C24. Other markers, such as GUS or molecular markers, on chromosome 2 may then be used to identify chromosomal regions which are derived from the Ler chromosome as a result of homeologous recombination. F2 plants of control transformants not expressing the antisense gene(s) can be analysed in parallel and the results can be used for comparison to homeologous recombination results obtained in antisense plants.

(ii) Transformation of C24 wildtvpe. hybrid plant production and analysis of homeoloeous meiotic recombination Introduction of MMR altering genes into wildtype C24 is done using the root transformation protocol as described in G(i) for transformation of hybrid plants.

Transformed plants are selected by resistance to either 10 mg/L hygromycin (in case of transformation with T-DNA's derived from pNOS-Hyg-SCV) or to 50 mg/L kanamycin (in case of transformation with T-DNA's derived from pBIN19).

Transgenic plants are then allowed to self-fertilise and to produce seeds (T1 seeds).

Segregation of the antibiotic resistance gene in the T1 population then indicates the number of transgene loci. Lines with a single transgene locus (indicated by a 3:1 ratio of resistant:sensitive plants) are selected and are bred to homozygosity. This is done by collecting selfed seeds (T2) from T1 plants and subsequent testing of at least four independent T2 seed populations for segregation of the antibiotic resistance gene. T2 populations which do not segregate the antibiotic resistance gene are assumed to be homozygous for both the resistance gene and the linked MMR altering gene.

C24 plants homozygous for the MMR altering gene are then crossed to Ler lines homozygous for recessive visual markers (see E(ii)) to produce C24/Ler hybrid plants as described in These F1 hybrids are then allowed to enter the reproductive phase and to produce gametes by meiotic division of microspore and megaspore mothercells. At meiosis, the DMC 1 promoter is activated and can direct the expression of antisense or sense genes described in and above, leading to decreased MSH6 and/or MSH3 gene expression. This in turn depletes the gamete mothercells of MSH6 and/or MSH3 9I 1 n AnAO PrCT/PO/0677 7717,,7 protein, thus causing alteration of MMR during meiotic divisions and increasing the recombination frequency between the homeologous chromosomes of C24 and Ler.

Recombination events are then scored in the F2 generation.

For recombination analysis, the hybrid plants are allowed to self-fertilise and to produce F2 seeds. F2 plants are then preselected for a first visual marker. Since this marker is recessive, its visual presence indicates homozygosity for Ler DNA at the relevant locus. Those F2 plants which show this first visual marker are then analysed for the presence or absence of a second visual marker which in the Ler parent is closely linked to the first marker. Absence of the second visual marker indicates recombination between the relevant C24 and Ler chromosomes between the first and second marker. The frequency of recombination in transgenic hybrids is compared to the recombination frequency in control hybrids not expressing the MMR altering gene.

Examples of recombination analysis are the following.

The Ler line NW22(disl. ga4 rhl) is used for crosses to transformedC24.

F2 plants are preselected first for thiamine requirement (thl) and then are further analysed for re-appearance of wildtype height (loss of ga4) and/or re-appearance of normal trichomes (loss of disl) as a result of recombination.

The Ler line NW10(rt-201, cer3 is used for crosses to transformedC24.

F2 plants are then preselected first for thiazole requirement (tz) and then are further analysed for re-appearance of normal, i.e. non-shiny stems (loss of cer3) as a result of recombination.

The Ler line NW134 (trg ga3 is used for crosses to transformedC24. F2 plants are first preselected for dwarfish appearance (ga3) and are then analysed for re-appearance of trichomes (loss of ttg) as a result of recombination.

Ler plants homozygous for abi3-1 and gll-1 are used for crosses to transformedC24.

F2 plants are first preselected for their ability to germinate in the presence of abscisic acid and are then analysed for re-appearance of trichomes on the leaves (loss of gll-1) as a result of recombination.

In the case of homeologous recombination between transformedC24 and the Ler line NW1 (an-1, py-1, gll-1. cer2-1, msl-1), recombination analysis is similar the one described above, except that the second marker is not a visual marker but has to be a molecular marker. This is because the Ler parent carries only one visual marker per chromosome.

I

TABLE 1: SSLP Markers in Arabidopsis thaliana Subspecies Chromosome RI Map PCR Primer Primer Sequence Tin I 0 C1 length/COL length/LER length/C24 12.3 ATEATI F GCCACTGCGTGAATGATATG 57.8 172 162 162 ATEATI R CGAACAGCCAACATTAATlCCC 58.2 I 9.3 NGA63 F AACCAAGGCACAGAAGCG 57.3 111 89 120 NGA63 R ACCCAAGTGATCGCCACC 59.6 1 40.1 NGA248 F TACCGAACCAAAACACAAAGG 56.1 143 129 no amplific.

181.2 NGAI128 F GGTCTGTTrGATGTrCGTAAGTCG 60.1 180 190 no aniplific.

1 81.2 NGA280 F CTGATCTCACGGACAATAGTGC 60.1 105 85 I111.4 NGAI1I F CTCCAGTTGGAAGCTAAAGGG 60 128 162 170 NGAI II R TGTTTTTTAGGACAAATGGCG 11 cca. 7.5 NGA 168 F CCTTICACA'TCCAAAACCCAC 578 23217 208 NGA 168 R GCACATACCCACAACCAGAkA

II

II

11

'I

11

'II

Ill 111 48 62.2 73 ca. 77 ca. 83 3.4 12.8 17.5 NGAI 126L NGA I 126R NGA361 L NGA361R NGA168 F NGA168 R AthBIO2 L AthBIO2 R AthUBIQUE

L

AMhBIQUE

R

NGA172

F

NGA172 R NGA126

F

NGA126

R

NGA162

F

NGA162

R

CGCTACGCTTTTCGGTAAAG 5 GCACAGTCCAAGTCACAACC 5

AAAGAGATGAGAATTTGGAC

ACATATCAATATATTAAAGTAGC

TCGTCTACTGCACTGCCG

GAGGACATGTATAGGAGCcTrCG

TGACCTCCTCTTCCATGGAG

TAACAGAAACCCAAAGCTTTC

AGGCAAATGTCCATTCATTG

ACGACATGGCAGAT~rCTCC AGCTGCT1TCCTTATAGCGTCC

CATCCGAATGCCATOTC

GAAAAAACGCTACTTUCGTGG

CAAGAGCAATATCAAGAGCAGC

CATGCAATTTGCATCTGAGG

CTCTGTCACTCTFITCCTCTGG

i7.8 i9.9 49.5 59.6 61.9 59.9 54.5 54. 1 57.8 55.4 56.1 58.2 55.8 60.1I 1914 151 141 146 162 119 107 199 120 135 209 148 136 147 89 196 ''4 135 139 148 140 no amplific.

no amplitic.

III

IV

IV

IV

V

V

V

V

81.8 19.8 24.1 102 11.8 16.7 19.9 20 NGA6 F NGA6 R NGA12 F NGA12 R NGA8 F NGA8 R NGAI1107 L NGA 1107 R NGA225 F NGA225 R NGA249 F NGA249 R CA72 F CA72 R NGA151 F NGA151 R TGGA11TICTTCCTCTCTTrCAC

ATGGAGAAGCTTACACTGATC

AATGTTGTCCTCCCCTCCTC

TGATGCTCTCTGAAACAAGAGC

GAGGGCAAATCTTTATTTCGG

TGGCTTTFCGTTTATAAACATCC

GCGAAAAAACAAAAAAATCCA

CGACGAATCGACAGAA'1TAGG

GAAATCCAAATCCCAGAGAGG

TCTCCCCACTAGTTTTGTGTCC

TACCGTCAATI'TCATCGCC

GGATCCCTAACTGTAAAATCCC

AATCCCAGTAACCAAACACACA

CCCAGTCTAACCACGACCAC

GTTGGGAAG1TTGCTGG

CAGTCTAAAAGCGAGAGTATGATG

56.1 56.1 59.9 58.2 56. 1 54.5 50. 2 58 58 60.1 55.4 58.2 56.3 61.9 55.8 58.6 143 123 247 234 154 198 150 140 119 189 125 115 124 110 F50 g1220 143 220 190 140 119 115 110 130 0 00 4 4 v

V

V

V

V

24 37.8 50 61.1 81.7 NGA 106 F NGA106

R

NGA139 F NGA139

R

NGA76 F NGA 76 R ATHS0191 L ATHS0191

R

NGAI129 F NGA129 R GTTATGjGAG'I1"IcFAGGGCACG

TGCCCCATVTGTTCTTCTC

AGAGCTACCAGATCCGATUG

GGTTTCG1TTCACTATCCAGG

GGAGAAAATGTCACTCTCCACC

AGGCATGG]GAGC

XATTTACG

CACACTGAAGAI'GGTCT-GAGG

55.9 59.9 157 123 174 132 231 250 148 156 177 179 I .(0 132 300 146 172 '~0 00 0 0%

-J

-4 WO 99/19492 WO 99/ 9492PCT[EP98/06977 SEQUENCE LISTING <110> <120> <130> <150> <151> <160> <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <223> <300> <301> <302> <303> <306> <307> <400> Rhone-Poulenc Agro; Betzner, Andreas Stefan; Doutriaux, Marie-Pascale; Freyssinec, Georges; Perez, Pascual.

Methods for obtaining plant varieties 3 95498C P09745 1997-10-10 98 1 23

DNA

Artificial sequence modified-base 14

I

modified-base 14

I

Degenerate oligonucleotides UPMU used to isolate AtMSH3 and AtMSH6.

Reenan and Kolodner Genetics 132 963- 973 1992 1 ggnccnaay atc! 23 ctggatccac n <210> 2 <211> 23 <212> DNA WO 99/19492 WO 99/ 9492PCT/EP98/06977 <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <223> 2 Artificial sequence modified-base

I

modified-base 18

I

Degenerate oligonucleotides DOMU used to isolate ACMSH-3 and AtMSH6.

<300> <301> Reenan and Kolodner <302> Genetics <303> 132 <306> 963-973 <307> 1992 <400> 2 ctggatccrt artgngtnrc raa <210> 3 <211> 24 <212> DNA <213> Artificial sequence <220> <223> MSH3 specific primer 636 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia <400> 3 tgctagtgcc tcttgcaagc tcat 24 <210> 4 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Primer AP1 for 2CR using cDNA of Arabidopsis thaliana ecotype Columbia containing adapter sequences ligated to both its ends <400> 4 WO 99/19492 ccatcctaat acgactcact atagggC PCT/EP98/06977 27 <210> <211> <212> <213> <220> <223> <400> 23

DNA

Artificial sequence Primer AP2 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia containing adapter sequences ligated to both its ends actcactata gggctcgagc ggc <210> <211> <212> <213> <220> <223> <400> 6

DNA

Artificial sequence MSH3 specific primer S525 for 2CR using cDNA of Arabidopsis thaliana ecotype Columbia 6 aggttctgat tatgtgtgac gctttactta <210> <211> <212> <213> <220> <223> <400> 7 29

DNA

Artificial sequence MSH3 specific primer S51 for 2CR using cDNA of Arabidopsis thaliana ecotype Columbia 7 ggatcgggta ctgggttttg agtgtgagg 29 <210> 8 <211> 24 <212> DNA <213> Artificial sequence <220> <223> MSH3 specific primer 635 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia WO 99/19492 WO 9919492PCTIEP98/06977 <400> 8 gcacgtgctt gatggtgtrt: ccac <210> <211> <212> <213> <220> <223> <400> 9 28

DNA

Artificial sequence MSH3 specific primer S523 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 9 tcagacagta tccagcatgg cagaagta 28 <210> <211> 33 <212> DNA <213> Artificial seauerice <220> <223> MSH3 specific primer 1S5 for 2CR using cDNA of Arabidopsis tha2.iana ecotype Columbia <400> atcccgggat gggcaagcaa aagcagcaga cga <210> 11 <211> 27 <212> DNA <213> Artificial sequence <220> <223> MSH3 specific primer S53 for 2CR using cDNA of Arabidopsis thaliana ecotype Columbia <400> 11 gacaaagagc gaaatgaggc cccttgg 27 <210> 12 <211> 1250 <212> DNA <213> Arabidopsis thaliana ecotype Columbia <223> Clone 52 WO 99/1 9492 PTE9/67 PCT/EP98/06977 <400> 12 cccgggatgg gcaagcaaaa gcagcagacg atctctcgtt tcttcgctcc caaacccaaa, tccccgactc gccactgtat tcacccaaaa caccaaagat tcatcatcga ccagatgtgg gagat cgcag agtgtgccaa attggtgtagr ggccctttc2t gatacaagtg tgtgt tgtgg gatgttagag ttcaatgata gagctgttgc ggacctacct gcagtagatg aaagaaatga atgaacatgc tttggatttg <210> <211> <212> <213> <220> <223> acgaaccgaa ccttctctcc agcctaaact ttctccagag ggaaatacac ttttgatggt cacgcgtgtt catttcgatt zgaagcagac zccggggacz: gtggttgtgg atgagagagt tcggtgttgt attctcatgag ttggccagcc caaacgttCcg aggttatttc agctggaggc cacatctgac tccggtagcc ttccaagcgt ttctcctcac atttctggaa accattggaa ggaagttggt gggtatttac gaatrttccat cgaaac:gca gzcggcg- t tggtgaagaa C aagtcggag tggcgt tgaa aagcggatta tctttcacaa agtggaacgt attatgtgaa tgctgaaaaa tgttcaagcc gaatcatcaa aagcttctct act caaaacc ccctcgccgg cagcaagtgg tacaggtaca gctcatatgg gtgagaagac ac cautaagt cataccaaag ggttttggtt acattaggct atttcgacag gaggctgtga caaacr-gaga gcctcactgg aaaat cagcg ggaatgtctt ctcgccctaa caccgccacc ccgaccacct cagtacccga aggaatatgt tggagctaaa gattcttcgg atcacaattt tggtgaacgc cccatggcgc ccacgcttga cacagagtaa gtggtattga gtgaagttgt ttttgagctt agtttttggt attgtttcag caggtaactt gcttgacagt cgctttgcca gaagatatcc cgccgccgcg tcccaattta C cccgaaacg gagcaagtac agaagacgcg catgacggcg aggatacaag aaaccggacc agcggctgag tttcttggtt aatgagtttt ttacgaagag gtcaccagct ggcacatgct caacggtaat agaagatgat tcatacaatt tctcaaacag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 aaaggatcct ttaccaaggg gcctcatttc gctctttgtc 1250 13 34

DNA

Artificial sequence MSH43 specific primer 2S5 for PCR using cDNA of ArabidopsiS thaliana ecotype Columbia WO 99/19492 WO 99/ 9492PC1'/EP98/06977 <400 atcccggg-c aaaatgaaca agctggt~tt agtc <210> <211> <212> <213> <220> <223> <400> 14 27

DNA

Artificial sequence MSH3 specific primer S52 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 14 gccacatctg actgttcaag ccctcgc <210> <211> 2110 <212> DNA <213> Arabidcosls :"aI.:ana ecotyoe Columbia <223> 'Clone 13 <400> gccacacctg tgaaaggatc tctctcagcc acctggctcc tagacactgg tgtt tctgag ggttgaagaa agtc-ttgaca tcggactgct gcaaattcag tgtgcgatct caatgccgga cgacatacta actgttcaag ctttaccaag aatactctgc ttat tccaca gtgactcatc atttctgctt ggttctgaga gctatgtcta aaagccacag cggc ttggca actcttttga aaacttctct atcacttcca ccctcgccct aacgttttgc cat--ctcaaac agcttggatt gggcctzcat aacagzcgga atatgaatca ctctatgcga gcatgggatc gagcaat tgt gatcatctga agttcattgc t aaagcaaga gaaaattgat ctgccctaaa gcgaccaatt tcgctctttg ggt tgtgaaa cacacttaca tagaaatttg tcatagttct atcacctgag tattcaacgt agttatggaa ctctgaaatg ttctgttatt taaggaagcg tcctgagctt E caagtaaca aar-aattcag cgcatatggtt atatctgctc tcccagctca ttttatctcg ggaataacaa gctattttac aggagtatgc tcatcccctg gctgttcgag gctgaagctc cagagatgac atggatcgga ccaggcttct ggcttgatgc gcagtgagtt tgctctcctc gaatctttca ttgcggggaa aatctgcaac t tgt ggt tga gtgacttgct gccaagcagt 120 180 240 300 360 420 480 540 600 660 720 780 840 tttagtcatc agggaaaagc tggattcctc gatagcttca tctcgcaaga agctcgctat WO 99/19492 WO 99/ 9492PCT/EP98/06977 tcgaaattt g t cccaaggtc ccccccagaa tgtgaaccga taaggctgcc t agaaacaag catacagtct tgacacaatt aggaaagagc ctttgtacca tgcttcagac acacataatc cactagcaca aaagagatgt attcccaggt cagttatgat gagctttggt catttcaatg gggagaacca gtttttaaag ttgacccggg gaattcttc cctatgaatt atagcagctg gcttcgtggg gttcaagctc aactatgtcc ggtcgtcatc t tgcatgcag tgctatatcc gcgtcattcg agcatccagc agaacctgct cacgacggtg tggttcttc cctgttggga catgatgatg C C Caagg C Cg gctgcaaaac gaaggacatg gcagacctga catgcttgga aagtgtcggg gggtgaaagt gcttggacga atagtttcct C.gctgcact gtcccgagt C ctgtactgga aaggggaata gtcaag-ttgc ccaagctgca atggcagaag CtCCgtrtC tagcca:rEgc ttgtcacgca cataccatgt cgacctacct ctcagcttgc tggaagccga aagaac cgag aatttgctct agattgctgg gatcacacat aaat agcacc gctagctcta caagagtt Cc ggactgtttg tgtggatgac gactatatta ttgccaaatt tttaatttcc cgtgcttgat t:acczttcta gc:ctatcat~a ctatgcaaca ttaccctgaa ctcgtatctg atataagctC ccagatacct ggtacgtgca aggcgcagaa ctctgaagag caaaatcaga ttgatagagc aagaagacta gcaactgaac agtagatact cactcccttt tgtgaaccag caagataact atcaccggac at aatggct c ggtgttttca gaagaattaa C Cagatgagc Ctacagcatc atagctgaga acatCgcaga gtgcgtggtc ccatcatgta agagagagaa gaatctatt gacccttgga ctaaaaccaa gcccgttga ttcgatatca accttgccar.

acacagattt caactctatc t Cgagataaa Ccgtcccaaa ctaacatggg aggttggttc ctcggatggg gtgaagcgtc ttggaagagg tcctagcaga tcagtaacgg aggataaagg tttgcagcag tacgtcgagc atacacgcat cggctctagg aagcattcga cttgttcatt 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2110 <210> <211> <212> <213> <220> <223> <400> 16 29

DNA

Artificial sequence MSH3 specific primer S51 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 16 WO 99/19492 WO 99/ 9492PCT/EP98/06977 8 ggaccgggta ccgggttttg agtgtgagg 2 <210> 17 <211> <212> DNA <213> Artificial sequence <220> <223> MSH3 specific primer S525 for 2CR using cDNA of Arabidoosis thaliana ecotype Columbia <400> 17 aggttctgat tatgtgtgac gctttactta 3 <210> <211>' <212> <213> <220> <221> <222> <223> <400> 18 3522

DNA

Arabidopsis chaliana ecotype Columbia CDs (100) .(3342) AtMSH3 full-length cDNA and deduced sequence of the encoded polypeptide 18 cctaagaaag cgcgcgaaaa ttggcaaccc aagttcgcca tagccacgac cacgacctc catc-ctctt aaacggagga gattacgaat aaagcaa-t atg Met

I

ggc aag caa Gly Lys Gin aag Lys 5 cag cag acg att Gin Gin Thr Ile tc Ser 10 cgt cc-c tcc gcc Arg Ph-e Phe Ala ccc aaa Pro Lys is 147 ccc aaa tcc Pro Lys Ser ccg cca ccg Pro Pro Pro aag ctt ctc Lys Leu Leu.

so ctt tc c ct Leu Ser Pro c cg Pro act cac gaa ccg Thr His Giu Pro aat As n 25 ccg gta gcc gaa Pro Val Ala Glu tca cca aca Ser Ser Thr 195 aag ata tcc gc Lys Ile Ser Ala ac t Thr gta tcc ttc tc cct tcc aag cgt Val Ser Phe Ser Pro Ser Lys Arg 243 291 tcc gac cac ctc gcc gac gcg cca Ser Asp His Leu Ala Ala Ala Ser 55 ccc Pro aaa aag cct aaa Lys Lys Pro Lys cac act caa His Thr Gin 70 aac cca gta ccc Asn Pro Val Pro ga t Asp 75 ccc aat tca cac Pro Asn Leu His ceia Gin 339 WO 99119492 WO 99/ 9492PCT/EP98/06977 aga ttt ctc cag Arg Phe Leu Gin aga Arg 8S ttt ctg gaa ccc Phe Leu Giu Pro tcg Se r 90 ecg gag gaa tat Pro Glu Giu Tyr gtt ccc Val Pro 387 gaa acg tea Glu Thr Ser gag cta aag Glu Leu Lys 115 tca Se r 1.00 tcg agg aaa cac Ser Arg Lys Tyr aca Thr 105 cca ttg gaa eag Pro Leu Glu Gin caa gt~g gt~g Gin Val Val 110 gaa gttc ggt Glu Val Giy 435 483 agc aag tac cea Ser Lys Tyr Pro gatc Asp 120 gtg gtt ttg at~g Vai Val Leu Met gtg Val1 125 tac agg Tyr Arg 130 tac aga tte ttc Tyr Arg Phe Phe gga Gly 135 gaa gac gcg gag Giu Asp Ala Giu ate Ile 140 gea gca cgc gt~g Ala Ala Arg Val ttcg Leu 145 ggt at~t tac get Gly Ile Tyr Ala cat His 150 at~g gatceae aat Met Asp His Asn ttc Phe 2.55 atg aeg geg agt Met Thr Ala Ser gtg Val 160 S31 579 627 eea aca ttt cga Pro Thr Phe Arg cttg Leu 1.65 aat. ttc ca-- gtg Asn Phe H Is Val aga Arcg 17C aga cga gt~g aat Arg Leu Val Asn gea gga Ala Gly 2.75 tac aag act.

Tyr Lys Ile eat ggc gca His Gly Ala 195 gg t Gly 180 gca gcg aag cag Val Vai Lys Gin act Thr 185 gaa act gea gee Giu Thr Ala Ala act aag tee Ile Lys Ser 190 tceg geg ttg Ser Ala Leu 675 723 aae egg ace gge Asn Arg Thr Gly ec Pro 200 ttt ttc egg gga Phe Phe Arg G 1 ctg Leu 205 tat ace Tyr Thr 210 aaa gee aeg ect Lys Ala Thr Leu gaa Giu 215 geg get gag gat Ala Ala G2.u Asp aca Ile 220 agt ggt ggt tgt Ser Gly Gly Cys 771 819 ggt Gly 225 ggt gaa gaa ggt Gly Glu Giu Gly ttt Phe 230 ggc tea cag agt Gly Ser Gin Ser aat Asn 235 ccc ttcg gtt tgt Phe Leu Val Cys gtt Val 240 gtg gat gag aga Val Asp Glu Arg gtt Val 245 aag teg gag aca Lys Ser Giu Thr tt~a Leu 250 gge tgt ggt att Gly Cys Gly Ile gaa at~g Giu Met 255 867 agt ttt gat Ser Phe Asp gaa. gtt gtt Glu Val Val 275 gtt Val1 260 aga gtce ggt gcc Arg Val Giy Val gt~t Val 265 gge gct gaa act.

Gly Val Glu Ile teg aca ggt Ser Thr Gly 270 agt gga tta Ser Gly Leu 915 963 tat gaa gag ttce Tyr Glu Glu Phe aat Asn 280 gat aac tte at~g Asp Asn Phe Met aga Arg 285 WO 99/19492 WO 99/ 9492PCT/EP98/06977 gag gct Gi'Lu Ala 290 gtg att ctg agc Val Ile Leu Ser t tg Leu 295 tca. cca. gct gag Ser Pro Ala Glu ccg Leu 300 trg ctt ggc cag Leu Leu Gly Gin cc,: Pro 305 ctrt tca caa caa Leu Ser Gin Gin act- Thr 310 gag aag ttt ttg Giu Lys Phe Leu gtg Val1 315 gca cat gct gga.

Ala Met Ala Gly cct Pro 320 1011 1059 1107 acc tca aac gtt Thr Ser Asn Val cga Arg 325 gtg gaa cgt gcc Val Glu Arg Ala tca Ser 330 ctg gat tgt ttc.

Leu Asp Cys Phe agc aat Ser Asn 335 ggt aat gca Gly Asn Ala ggt aac tta.

Gly Asn Leu 355 gta Val1 340 gat gag gtt att Asp Giu Val Ile tca Ser 345 tta tgt gaa aaa Leu Cys Glu Lys atc agc gca Ile Ser Ala 350 gct gaa aaa Ala Glu Lys 1155 1203 gaa gat gat aaa Giu Asp Asp Lys gaa Glu 360 atg aag ctg gag Met Lys Leu Glu gct Ala 365 gga atg Gl1y Me: 370 tct tgc ttg aca Ser Cys Leu Thr gtt Val1 375 cact aca. att atg His Thr Ile Met aac Asn 380 atg cca cat ctg Met Pro His Leu act Thr 385 gzc caa. gcc ctc Val. Gin Ala Leu gcc Ala 390 cta acg t-tt tgc Leu Th-r Phe Cys c--c aaa cag Ltt Leu Lys Gin Phe gga Gly 400 1251 1299 1347 ttt gaa agg atc Phe Glu Arg Ile ctt Leu tac caa ggg gcc Tyr Gin Gly Ala tca Ser 410 ttt cgc tc t ttg Phe Arg Ser Leu tca agt Ser Ser 415 aac aca gag Asn Thr Glu gtg aaa aat Val Lys Asn 435 atg Met 420 act ctc tca grcc Thr Leu Ser Ala aat Asn 425 act ccg caa cag Thr Leu GIn Gin ttg gag gt Leu Glu Val 430 ttc cat aat Phe His Asn 1395 1443 aat tca. gat gga Asn Ser Asp Gly Ccg Ser 440 gaa tct ggc tcc Giu Ser Gly Ser tta Leu 445 atg aat Met Asn 450 cac aca. ctt aca His Thr Leu Thr gta Val 455 tat gct tcc agg Tyr Gly Ser Arg ctt Leu 460 ctt aga cac tgg Leu Arg His Trp gtg Val 465 act cat cct ct.

Thr His Pro Leu tgc Cys 470 gat aga aat ttg Asp Arg Asn Leu aca Ile 475 tct gct cgg ctt Ser Ala Arg Leu gat Asp 480 1491.

1539 1587 gct gtt tct. gag Ala Val Ser Glu att Ile 485 tc gct tgc atg Ser Ala Cys Met gga tc Giy Ser 490 cat agt tct His Ser Ser tcc cag Ser Gin 49S WO 99/1 9492 PTE9/67 PCT/EP98/06977 ccc agc agt Leu Ser Ser cct gag ctt Pro Glu Phe 515 gag Giu 500 ttg gct gaa gaa Leu Val Giu Giu ggt Gly 505 tct gag aga gca Ser Giu Arg Ala att gta tca Ile Val Ser 510 atg tct aga Met Ser Arg 1635 1683 tat ctc gtg ctc Tyr Leu Val Leu tcc Ser 520 tca gtc ttg aca Ser Val Leu Thr gct Ala 525 tca tct Ser Ser gat act caa cgt Asp Ile Gin Arg gga Gly 535 ata aca aga atc Ile Thr Arg Ile ttt Phe 540 cat cgg act gct His Arg Thr Ala aaa Lys 545 gcc aca. gag ttc Ala Thr Giu Phe act Ile 550 gca gtt atg gaa Ala Val Met Glu att tta ctt gcg Ile Leu Leu Ala ggg Gly 560 1731 1779 1827 aag caa att cag Lys Gin Ilie Gin cgg Arg 565 ctt ggc ata aag Leu Gly Ile Lys c aa Gin 570 gac tct gaa atg Asp Ser Giu Met agg agt Arg Ser 575 atg caa tct.

Met Gin Ser gtt act tca Vai Ile Ser 595 gca Al a 580 act gtg cga tc: Thr Val Arg Ser act Th r 585 ctt ttg aga aaa Leu Leu Arg Lys ttg act tct Leu Ile Ser 590 ctt ctc tct Leu Leu Ser 1875 1923 tcc ccz gtt gtg Ser Pro Val Val gct Val1 600 gac aat gcc gga Asp Asn Ala Gly aaa Lys 605 gcc cta Ala Leu 610 aat aag gaa gcg Asn Lys Giu Ala gct Ala 615 gtt cga ggt gac Val Arg Gly Asp ttg Leu 620 ctc gac aca cta Leu Asp Ile Leu ac lie 625 act ccc agc: gac Thr Ser Ser Asp caa Gin 630 ttt oct gag ctt Phe Pro Giu Leu gaa. gct cgc caa Giu Ala Arg Gin gca Ala 640 1971 2019 2067 gtt tta gtc atc Val Leu Val Ilie agg Arg 645 gaa aag ctg gat Giu Lys Leu Asp tcc Ser 650 tcg ata gct tca Ser Ile Ala Ser ttt cgc Phe Arg 655 aag aag ctc Lys Lys Leu aca cat tog Thr His Leu 675 gct Ala 660 act cga aat ttg Ile Arg Asn Leu gaa Giu 665 ttt ctt caa. gtg Phe Leu Gin Val tcg ggg atc Ser Gly Ile 670 211S aca. gag ctg ccc Ilie Giu Leu Pro gtt Val 680 gat tcc aag gtc cct atg aat tgg Asp Ser Lys Val Pro His Asn Trp 685 2163 gtg aaa Val Lys 690 gta aat. agc acc Val Asn Ser Thr aag Lys 695 aag act att cga, Lys Thr Ile Arg tat Tyr 700 cat ccc cca. gaa.

His Pro Pro Giu 2211 WO 99/19492 WO 9919492PCT/EP98/06977 ata Ile 705 gta gct ggc ttg Val Ala Gly Leu gatc Asp 710 gag cca gct clca Glu Leu Ala Leu gca Ala 715 act gaa cat ctc Thr Glu His Leu gcc Ala 720 2259 2307 att gtg aac cga Ile Val Asn Arg gcc Ala 725 tcg cgg gat agt Ser Trp Asip Ser tcc Phe 730 ctc aag agt tc Leu Lys Ser Phe agt aga Ser Arg 735 tac cac aca Tyr Tyr Thr tgt tcg cac Cys Leu His 755 gat Asp 740 ttc aag gcc gcc Phe Lys Ala Ala gcc Val 745 caa gcc ctt gct G lm Ala Leu Ala gca ccg gac Ala Leu Asp 750 tat gcc cgc Tyr Val Arg 2355 2403 ccc ccc tca act Ser Leu Ser Thr cta Leu 760 tc aga. aac aag Ser Arg Asn Lys aac Asn 765 ccc gag Pro Giu 770 ccc gcg gat gac Phe Val Asp Asp cgt Cys 775 gaa cca. gtt gag Glu Pro Vai Glu aca Ile 780 aac ata cag cc Asn Ile Gin Ser gc Gly 785 cgt cat ccc gta Arg His Pro Val ca Le u 790 gag ac-- ata :ca G K- T-r 1le Leu caa Gin 795 gaz aac cc-c gc Aso Asn Phe Val cca Pro 800 2451 2499 2547 aac gac aca at Asn Asp Thr Ile ttg Leu 805 cat gca gaa ggg His Ala Gly aa G 1u 810 tat tgc caa at Tyr Cys Gin Ile atc acc Ile Thr 815 gga ccc aac Gly Pro Asn act tcc ata Ile Ser Ile 835 atg Met 820 gga gga aag agc Gly Giy Lys Ser c-gc Cys 825 tat acc cgt caa Tyr Ile Arg Gin gct gct tta Val Ala Leu 830 tca tcc gcc Ser Phe Ala 2595 2643 acg gcc- cag gcc Met Ala Gin Val gg c Giv 840 tcc ccc. gta cca Ser Phe Vai Pro gcg Ala 845 aag ct~g Lys Leu 850 cac gt~g ccc gac His Val Leu Asp ggc Gly 855 gcc ccc acc cgg Vai Phe Thr Arg acg Met 860 ggt gct tca. gac Gly Ala Ser Asp agt Ser 865 acc cag cac ggc Ile Gin His Gly aga.

Arg 870 agt acc ccc cca Ser Thr Phe Leu gaa Glu 875 gaa. tca agc gaa Giu Leu Ser Glu gcg Al a 880 2691 2739 2787 tca cac aca atc Ser His Ile Ile aga Arg 885 acc tgt tc tc Thr Cys Ser Ser cgt Arg 890 tcg ccc gt: aca Ser Leu Val Ile cta. gat Leu Asp 895 gag ctc gga Glu Leu Gly aga Arg 900 ggc act agc aca Gly Thr Ser Thr cac His 905 gac ggc gta gcc Asp Giy Vai Ala act gcc cat Ile Ala Tyr 910 2835 WO 99/1 9492 PTE9/67 PCTIEP98/06977 13 gca aca cca cag cat ctc cca gca gaa aag aga cgc ccg gtt ccc ccc Ala Thr Leu Gin His Leu Leu Ala Giu Lys Arg Cys Leu Val Leu Phe 915 920 925 2883 gtc acg Val Thr 930 tc gtt Ser Vai 945 cat tac cct gaa aca gct His Tyr Pro Glu Ile Ala 935 ggg aca cac cat gtc tcg Gly Thr Tyr His Val Ser 950 ggc agt tat gat cat gat gat gtg Gly Ser Tyr Asp His Asp Asp Val 965 ggt ctt tgc agc agg agc ccc. ggt Gly Leu Cys Ser Arg Ser Phe Gly 980 aca cct cca tca cgt ata cgt cga lie Pro Pro Ser Cys ile Arg Ara 995 1000 gag acc agt aac gga tcc cca ggt Glu Ile Ser Asn Gly Phe Pro Gly 940 tat ccg aca ttg cag aag gat aaa Tyr Leu Thr Leu Gin Lys Asp Lys 955 960 acc tac cta tat aag ctt gcg cgt Thr Tyr Leu Tyr Lys Leu Val Arg 970 975 ccc aag gcc gct cag ccc gcc cag Phe Lys Val Ala Gin Leu Ala Gin 985 990 gcc act tca acg gct gca aaa ctg Ala Ile Ser Mec Ala Ala Lys Leu 1005 aga aac- aca cgc acg gga gaa cca Arg Asn Thr Arg Met Gly Gl1u Pro 1020 gca gaa gaa tc act tcg gcc. cta Ala Glu Glu Ser Ile Ser Ala Leu 1035 1040 2931 2979 gaa gct Glu Ala 1010 gaa gga Glu Gly 1025 gag gca cgt gca aga gag Glu Val Arg Ala Arg Glu 1015 cac gaa gaa ccg aga ggc His Glu Glu Pro Arg Gly 1030 3027 3075 3123 3171 3219 3267 3315 3362 3422 3482 3522 ggt gac ctg ccc gca gac ctg aaa ccc gcc ccc cc gaa gag gac ccc Gly Asp Leu Phe Ala Asp Leu Lys Phe Ala Leu Ser Glu Glu Asp Pro 1045 1050 1055 tgg aaa gca ccc gag ccc tta aag cat gct tgg aag act gct ggc aaa Trp Lys Ala Phe Giu Phe Leu Lys His Ala Trp Lys Ile Ala Gly Lys 1060 1065 1070 acc aga cta aaa cca act tgt cca. ccc tgacttaacc tc-aacatcat Ile Arg Leu Lys Pro Thr Cys Ser Phe 1075 1080 agcaactgca aggtcccgat catccgtcag ccgcgcacca acccatgcgc accagtacaa caagaaaaga gaactagaga gacggatcct aacccggtgc cgcagcacac ctttccccca cccgcataaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa <210> <211> c2 12 19 1082.

PRT

WO 99/19492 PCTIEP98/06977 <213> <223> <400> Met Gly

I

Pro Lys Pro Pro Lys Leu Leu Ser Arg Phe G1 u Thr Glu Leu Tyr Arg 230 Leu Gly 145 Pro Thr Tyr Lys His Gly Tyr Thr 210 Gly Gly 225 Arabidopsis thaliana ecotype Columbia Polypeptide MSH3 19 Lys Ser Pro Leu Pro Leu Ser Lys 115 Tyr lie Phe Ile Ala 195 Lys Glu Gin Pro Lys Ser His Gln Ser 100 Ser Arg Tyr Arg Gly 280 Asn Ala Glu Lys 5 Thr Ile Asp Thr Arg Ser Lys Phe Ala Leu 265 Val Arg Thr Gl Gin His Ser His Gln 70 Phe Arg Tyr Phe His 150 Asn Val Thr Leu Phe Gln Glu Ala Leu 55 Asn Leu Lv s Pro Gly 135 Met Phe Lys Gly Glu 215 Gly Pro Thr 40 Ala Pro Ty r Asp 1220 GIu Asp H s Gin Pro 200 Ala Ser Ile Asn 25 Va1 Ala Val Pro Thr 205 Val Asp His Va2 Thr 185 Phe Ala Gln Ser 10 Pro Ser Ala Pro Ser 90 Pro Val Ala Asn Arg 170 Glu Phe Glu Ser Arg Val Phe Ser Asp 75 Pro Leu Leu Glu Phe i55 Arg Thr Arg Asp Asn 235 Phe Ala Ser Pro Pro Glu Met lie 140 Met Leu Ala Gly lie 220 Phe Phe Glu Pro Lys Asn Glu G1 n Val 125 Ala Thr Val Ala Leu 205 Ser Leu Ala Ser Ser Lys Leu Tyr Gin 110 Glu Ala Ala Asn Ile 190 Ser Gly Val Pro Ser Lys Pro His Va2 Val Va2 Arg Ser Ala 275 Lys Ala Gly Cys Lys Thr Arg Lys Gln Pro Va1 Gly Val Val 160 Gly Ser Leu Cys Val 240 230 Val Asp Glu Arg Val Lys Ser Glu Thr Leu Gly Cys Gly Ile Glu Met 255 245 250 WO 99/19492 WO 99/ 9492PCT/EP98/06977 Ser Phe Asp Val Arg Val Gly Val Val Gly Val Glu Ile Ser Thr Gly 260 265 270 Glu Val Val Tyr Glu Glu Phe Asn Asp Asn Phe Met Arg Ser Gly Leu 275 280 285 Glu Ala Val Ile Leu Ser Leu Ser Pro Ala Giu Leu Leu Leu Giy Gln 290 295 300 Pro Leu Ser Gin Gin Thr Giu Lys Phe Leu Val Ala Met Ala Giy Pro 305 310 315 320 Thr Ser Asn Val Arg Val Giu Arg Ala Ser Leu Asp Cys Phe Ser Asn 325 330 335 Gly Asn Ala Val Asp Glu Val Ilie Ser Leu Cys Giu Lys Ile Ser Ala 340 345 350 Gly Asn Leu Glu Asp Asp Lys Giu Met Lys Leu Glu Ala Ala Glu Lys 355 360 365 Gly Met Ser Cvs Leu Thr Val His T-.r I I -e Met Asn Met Pro His Leu 370 375 380 Thr Val Gin Ala Leu Ala Leu Tlhr Phe Cvs His Leu Lys Gin Phe Gly 385 390 395 400 Phe Glu Arg Ile Leu Tyr Gin Giy Ala Ser Phe Arg Ser Leu Ser Ser 405 410 415 Asn Thr Giu Met Thr Leu Ser Ala Asn Thr Leu Gin Gin Leu Giu Val 420 425 430 Val Lys Asn Asn Ser Asp Gly Ser Glu Ser Gly Ser Leu Phe His Asfl 435 440 445 Met Asn His Thr Leu Thr Val Tyr Gly Ser Arg Leu Leu Arg His Trp 450 455 460 Val Thr His Pro Leu Cys Asp Arg Asn Leu Ilie Ser Ala Arg Leu Asp 465 470 475 480 Ala Val Ser Giu Ile Ser Ala Cys Met Gly Ser His Ser Ser Ser Gin 485 490 495 Leu Ser Ser Giu Leu Val Glu Glu Gly Ser Glu Arg Ala Ile Val Ser 500 505 510 Pro Giu Phe Tyr Leu Val Leu Ser Ser Val Leu Thr Ala Met Ser Arg 515 520 525 Ser Ser Asp Ile Gin Arg Gly Ile Thr Arg Ile Phe His Arg Thr Ala 530 535 540 WO 99/19492 WO 99/ 9492PCT/EP98/06977 Lys 545 Lys Met Val Ala Ile 625 Val1 Lys T17r Val1 Ile 705 Ile Tyr Cys Pro Gly 785 Asn Ala Gi1nr Gin Ile Leu 610 Thr Leu Lys His Lys 690 Val1 Val1 Tyr Leu Giu 770 Arg Asp Thr Ile Ser Ser 595 As n Ser Val1 Le u Leu 675 Val1 Ala Asn Thr His 755 Phe His Thr Giu Phe Ile 550 Gin Arg Leu 565 Aia Thr Val 580 Ser Pro Val Lys Giu Aia Ser Asp Gin 630 Ile Arg Glu 645 Ala Ile Arg 660 Ile Glu Leu Asn Ser Thr Gly Leu Asp 710 Arg Ala Ser 725 Asp Phe Lys 740 Ser Leu Ser Val Asp Asp Pro Vai Leu 790 Ile Leu His 805 Ala Vai Met Giu Ala Ile Leu Leu Ala Gly Gly Arg Val1 Ala 615 Phe Lys Asn Pro Lys 695 Giu Trp Ala Thr Cys 775 Giu Ala I Ie Ser Val1 600 Val1 Pro Val 680 Lys Leu Aso Al a Leu 760 Glu Thr Glu Lys Thr 585 Asp Arg Giu Asp Gi 1u 665 Asp Thr Al a Se r Val1 745 Ser Pro Ile Gly Gin 570 Leu Asn Gly Leu Ser 650 Phe Ser Ile Leu Phe 730 Gin Arg Val Leu Glu 810 555 Asp Leu Ala Asp Ala 635 Ser Leu Lys Arg Al a 715 Leu Ala Asn Giu Gin 795 'Tyr Ser Arg Gly Leu 620 Giu Ile Gin Val Tyr 700 Thr Lys Leu Lys Ile 780 Asp Cys Giu Lys Lys 605 Leu Ala Ala Val1 Pro 685 His Giu Ser Ala Asn 765 Asn Asn Gin Met Arg 575 Leu Ile 590 Leu Leu Asp Ile Arg Gin Ser Phe Ser Gly 670 His Asn Pro Pro His Leu Phe Ser 735 Ala Leu 750 Tyr Val Ile Gin Phe Val Ile Ile 815 560 Se r Ser Ser Leu Al a 640 Arg Ile Trp Glu Al a 720 Arg Asp Arg Ser Pro 800 Thr Gly Pro Asn Met Gly Gly Lys Ser Cys Tyr Ilie Arg Gin Val Ala Leu 820 825 830 WO 99/19492 WO 99/ 9492PCT/EP98/06977 Ile Ser I 8 Lys Leu H 850 Ser Ile G 865 Ser H-isI Glu LeuC Ala Thr Val Thr 930 Ser Val 945 Gly Ser Gly Leu Ile Pro Glu Ala 1010 Glu Gly 1025 Gly Asp Trp Lys le 35 i2s lin :le ;ly 4 eu 915 His Gly Tyr CyE P rc 99E Gl' Hi~ Le' Al Met Ala Gin Vai Gly E 840 Val Leu Asp Gly Vai 855 His Gly Arg Ser Thr 870 Ile Arg Thr Cys Ser 885 Arg Gly Thr Ser Thr 900 Gin His Leu Leu Ala 920 Tyr Pro Giu Ile Ala 935 Thr Tyr His Val Ser 950 Asp His Asp Asp Val 965 Ser Arg Ser Phe Gly 980 Ser Cys Ile Arg Arg 1000 .i Val Arg Ala Arg Glu 1015 sGlu Giu Pro Arg Gly 1030 u Phe Ala Asp Leu Lys 1045 a Phe Giu Phe Leu~ Lys 1060 ;er Phe Val Pro Ala Ser Phe Ala 845 Phe Phe Ser His 905 Glu Glu Tvr Thr Phe 985 Ala Arc Al PhE Thr Arg Met: C 860 Leu Giu Giu 1 875 Arg Ser Leu 890 Asp Gly Val Lys Arg Cys Ile Ser Asn 940 Leu Thr Le,,: 955 Tyr ileu Tyr 9,70 Lys Val Ala Ile Ser Met: As-n Thir Arg 1020 SGlu Glu Ser 1035 SAla Leu Ser 1050 ;iy Ala Ser Asp ~eu lal kl a Leu 925 Gly Gin Lys Gin Ala .00S Met IlE Gli Ser Giu Ala 880 Ile Leu Asp 895 Ile Ala Tyr 910 Val Leu Phe Phe Pro Gly Lys Asp Lys 960 Leu Val Arg 975 Leu Ala Gin 990 *Ala Lys Leu *Gly Glu Pro Ser Ala Leu 1040 i Giu Asp Pro 1055 e Ala Gly Lys 1070 His Ala Trp Lys tIl 1065 Ilie Arg Leu Lys Pro Thr Cys Ser Phe 1075 1080 <210> <211> <212> <213> 24

DN~A

Artificial sequence '6 WO 99/1 9492 PCT/EP98/06977 <220> <223> <400> MSH6 specific primer 638 for 2CR using cDNA of Arabidopsis thaliana ecotype Columbia tctctaccag gcgacgaaaa accg <210> <211> <212> <213> <220> <223> <400> 21 28

DNA

Artificial sequence Primer S81 for 2CR using cDNA of Arabidopsis thaliana ecotype Col1umb ia 21 cgtcgccztt agcatcccct tccttcac 2 <210> 22 <211> <212> DNA <213> Artificial sequence <220> <223> MSH6 specific primer S823 for PCR usi-ng cDNA of Arabidopsis thaliana ecotype Columbia <400> 22 gcttggcgca tctaatagaa tcatgacagg 3 <210> 23 <211> 24 <212> DNA <213> Artificial sequence <220> <223> MSH6 specific primer 637 for 2CR using cDNA of Arabidopsis thaliana ecotype Columbia <400> 23 gacagcgtca gttcttcaga atgc 2 <210> 24 <211> 33 <212> DNA WO 99/1 9492 PTE9/67 PCT/EP98/06977 19 <213> Artificial sequence ,-223; MSH6 specific primer 1S8 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia <:400> 24 atcccgggat gcagcgccag agaccgatt tgt 33 <:210> <211> 27 <212> DNA <213> Artificial sequence <220> <223> MSI46 specific primer S83 for 2CR using cDNA of Arabidopsis thaliana ecotype Columbia <:400> cgcza:c!:a: ggczqc:zccq aatcga 27 <210> <211> <212> <213> <223> <400> cccgggatgc acgaagggtt rttaatgtga tctgtcgatg ccgtctggat atgcataagt gttgttccgc cgttccaata gaacctagat gggatgcgtc gaggacaagg 26 1385

DNA

Arabidoosis thaliana ecotype Columbia Clone 43 26 agcgccagag tggtttccgg aggaagggga aggt tagagg t taagccggc ttgtaaaagt tgaatgattc atggtaaaac cagtagaaga cacgtgcttC ttcctgtatt atcgattztg tctttcttcc cgatgctgct agcggcgggg tgctaaaggc gacgcttctg aacggatact ccaccggaga tgaatccgcc ggtgatgctt cgatgatcga gattgtcg atctctatgt atgaaggcta tcaagaaaga aaccatgctt tataggagta gatggcgatg tcgcttgaag cgagttctgg ggactccaac aaaaggctga aaaaacccac ggcggcgact gcggcagcgg aggaccacga tacgttttgc tgtttcgaaa aggttccgcg tcgtgtcctg cgtccctgtt ctccaatatt gagagaggag ccgagaagat atgatgttat tcctcaattt ttagtttcag tgggagagct ttcctggtcc agaaacacca aggatgaaat gactttaag aaatgctcca ggatccggtt 120 180 240 300 360 420 480 540 600 660 WO 99/19492 WO 9919492PCT/EP98/06977 cg ggagaga atcagggatg ataccacctg agtgaatata ctagatgcgg aaatgcagac gctcgtggat agaggtgcta agcgagggaa gagctacaaa ~tttgggt:zg caggr-tcc gctctaagga gtaatggggg aaaggctctt cttagtgctc aagcacgggg acgatggtaa tacaaatatc tgccatccac acggcaaact gaaagac tgc cttctgggga ttcagaagtg ctttataaac agaaagaagt ccaatagaag atgttttcaa tggacat tgt aattaggtca aggttggtat ataaagt tgg atactataat acatcgggcc agtgttcaac ggtccatcag caaaggaagc aatatacgct atacagatgc ctgaatcatg t tggagagct atatttttcc atcttgagat t tgataactg tcaaagatgt cagaaagtat t cggacgcat aaaaagtgct gaat tgatct t ctgtaaact aaacgaagga acgtcccgat gaaaatgtct caaggagctt ctctgaaagt acgaat cgagr tccaaggaag tgatgccgtc tgcatatggacgacgazcca gttaza--=ac gacagggticr tgctggagtt gaactgtrct aattaatcat ataccaaqt atttaacaat tgttagtcca agaaagcat c gcaaatcact caagtctagc gaaacaacga gttgttggct tcctatatta accaaattg gatcc-Ccttt gcatcacaaa aaagtgggga gactggaaga gggatagatg cagctagaaa ctagttcagg cacctcttg zcatatgccg agcaaagggc acggcggtac agaaatataa gttgatggc ctgtctaggc tacaggggtt agc tgtgatg actggtaagc aataaacggc ggccagtat c gttcgatcat gttaaagcat ctacagaagg aatggcttga acgatagaaa agcaatattg aattttatga tgaccatgag aggcagtgca catctgacca tattaactcc ctataaaaga ttgactgtgc CtCE tggagc tat caagaga agttggctcc tagaatctaa C aaatgaatg taaagctaga gtcccagaat gcggtccttc gactcttaag ttgatgtagt tccacaaact cagcctctgt ttgggcaaat aatcaaatat gtcttctcga gaccttacac gagtgttaag gctgtatgag tggtgtggga aaagctatta agcaaaagcc atcaacagca gatcaaaatg tgccttgagg gttattgacg agcacaaaag agtaccacaa cggatacttt tgatgttgcc agatgtactt tgatggccag agggaccttg gaattggatc tgaagaattc tccagactta gttgcctgct tgtgaaaggg gatgagtttg 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2188 gtaggaaaaa gcgggctaga gttatttctt tctcaattcg aagcagccat agatagcg WO 99/1 9492 PTE9/67 PCT/EP98/06977 <210> <211> <212> <213> <223> <400> catcagcctc 27 1385

DNA

Arabidopsis chaliana ecotype Columbia Clone 62 27 tgtgttgcct gctcttctgg ggaaaaaagt gctgaaacaa cgagttaaag catttgggca aggaatcaaa aaagcgggc t attatcagaa c-.tttatcga tcctcgagatc t t t-tcccga tccaaggact atatactcct tgacgggacc tctttgccca ctatcttcac tagaatgcac ttgacgaact ttcgtcacct t caccaagga aatcaagatc taaccgaggg caaaccaagt aaaacttcaa agtcattggt aattgtgaaa gggttcagaa tatgatgagt agagttattt ccaagatgtg aagagcaact ttttgcaatc aucagaagct acggcatcca tggcgaggc t aaac at ggg c acttggctgc aaggc ttggc tgagacagcg gggcagagga ggtagagaaa attcqcgtct tgattatcaa agcttgtcct ggttgaaaca gtcaagtgag gggtatttct ttgctttata ctttctcaat acagatgaaa caatggtctg gcagcaagtc acagazcaga tttgcagttg agaagaagca ggaaaatcaa tacgtgccgt gcatctgata tcagttcttc actagtactt gttcaatgtc cacccacgtg ccacgtggtt gagagctacg gcatcaggtg ctaagatctg cgagtcgccc gtggaattga aactctgtaa t cgaagcagc acgctgaaac aggtcattca tctctgctgg at.cagaaaac cagccgatgg gtggcagcat CtcttCttcg gtgagtcttg gaatcacgac agaatgcaac tcgatggata ggatgctctt tcacctcgaa gtgatcaaga gacttcaagt ctgctcaagc agttctcaag acaacaatgc tctgttgttg acttcctata catagatagc tctcacaata caccataagc aagcatggcc aaaagggcca tcaattgcct tcatcctcgg tgcaacatgt cgaaatctcc aggagagagt tcaggattca cgccattgca tgcaacacat acacatggct cctagtgttc ggcactcatg catgaagaga t ctgcatgaa ccc cat tggc gctctacaga t tagtaggaa gactttccaa cttatcgaac tgcctagatg aggcctgtca acactcaaaa gtctccgaatg tcattgttac ctggccgtta ctcgtggata acctttttgg ctagtaatcc tactcggttt taccaccctc tgcgcattca ttgtaccgtt gctggaatac tcaattgggg gactggctca gaagatgact 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 acgacacttt gttttgctta tggcatgaga tcaaatcctc ttactgtgtt cccaaataac WO 99/19492 WO 99/ 9492PCT/EP98/06977 CCggg 1385 <210> 28 <211> 34 <212> DNA <213> Artificial sequence <220> <223> MSH6 specific primer 2S8 for PCR using cDNA of Arabidopsis thaJliana ecotype Columbia <400> 26 atcccgggtt atttgggaac acagtaagag gact.

<210> 29 <211> 2-7 <212> DNA <213> Artificial sequence <220> <223> MSH6 specific primer S82 fcr 2CR using cDNA of Arabidopsis thaliana ecotype Columbia <400> 29 gcgttcgatc atcagcctct gtgttgc <210> <211> 3606 <212> DNA <213> Arabidopsis thaliana ecotype Columbia <220> <221> <222> <223> <400>

CDS

(142) (3468) AtMSH6 full-length cDNA and deduced sequence of the encoded polypeptide aaaagttgag ccctgaggag tatcgtttcc gccatttcta cgacgcaagg cgaaaatttt tggcgccaat ctttcccccc tttcgaattc tctcagctca aaacatcgtt tctctctcac 120 tctctctcac aattccaaaa a atg cag cgc cag aga tcg att ttg tct ttc Met Gin Arg Gin Arg Ser Ile Leu Ser Phe 1 S WO 99/19492 WO 99/ 9492PCT/EP98/06977 ttc caa aaa Phe Gin Lys got got agc Al.a Ala Ser gaa ggg gat Glu Gly Asp ccc acc Pro Thr is gcg gcg act Ala Ala Thr acg aag ggt Thr Lys Gly 20 ttg gtt tc Leu Val Ser ggc gat Gly Asp ggc Gly ggg ggc ggc ago Gly Gly Gly Ser gga Gly 35 gga cca oga ttt Gly Pro Arg Phe aat gtg aag Asn Val Arg gtt tog aaa Val Ser Lys 219 267 315 gct aaa ggc gac Ala Lys Gly Asp got Ala 50 tct gta cgt ttt Ser Val Arg Phe got Ala tot gtc Ser Val gat gag gtt aga Asp Glu Val Arg gga Gly 65 acg gat act coa Thr Asp Thr Pro oog Pro gag aag gtt oog Giu Lys Val Pro ogt Arg ogt gto otg oog Arg Val Leu Pro tot Ser 80 gga ttt aag oog Gly Phe Lys Pro got Ala 85 gaa too goo gst Glu Ser Ala Gly gat Asp 363 411 459 507 gct tcg too ctg Ala Ser Ser Leu ttc Phe tcc aac ata Ser Asn 71e Met cat His 2.00 aag ttt gta aaa Lys Phe Val Lys gtc gat Val Asp 105 cog ctg Pro Leu gat cga gat Asp Arg Asp aat gat tca Asn Asp Ser 125 tgt Cys 110 tot gga gag agg Ser Gly Glu Arg ago Ser 115 oga gaa gat gtt Arg Glu Asp Val gt t Val 120 tot cta tgt atg Ser Leu Cys Met a ag Lys 130 got aat gat gt Ala Asn Asp Val att Ile 135 ccot caa ttt Pro Gin Phe 555 cgt too Arg Ser 140 aat aat ggt aaa Asn Asn Gly Lys act Thr 145 caa gaa aga aac Gin Giu Arg Asn cat His 150 got ttt, agt ttC Ala Phe Ser Phe ag t Ser 155 ggg aga. got gaa Gly Arg Ala Giu Ott Leu 160 aga tca gta gaa Arg Ser Val Glu ga t Asp 165 ata gga gta. gat Ile Gly Val Asp ggo Gly 170 603 651 699 gat gtt cot ggt Asp Val Pro Gly oca Pro 175 gaa aca oca ggg Giu Thr Pro Gly at~g Met 180 ogt oca ogt got, Arg Pro Arg Ala tot. ogo Ser Arg 185 ttg aag oga Leu Lys Arg cot gta ttg Pro Val Leu 205 gtt Val 190 otg gag gat gaa Leu Giu Asp Giu atg Met 195 act ttt aag Thr Phe Lys gag gat aag gtt Giu Asp Lys Val 200 cag gat cog gtt Gin Asp Pro Val 215 747 795 gao tot aac aaa Asp Ser Asn Lys agg Arg 210 ctg aaa atg oto Leu Lys Met Leu WO 99/19492 WO 99/ 9492PCT/EP98/06977 tcgt gga Cys Gi y 220 gag aag aaa gaa Glu Lys Lys Giu gta Val1 225 aac gaa gga acc: Asn Glu Gly Thr aaa Lys 230 ttt gaa tgg ctt Phe Glu Trp Leu gag Giu 235 ct t: cga ac Ser Ser Arg Ile agg Arg 240 gat gcc aat aga Asp Ala Asn Arg aga Arg 245 cgt cct gat gat Arg Pro Asp Asp ccc Pro 250 843 891 939 ctt tac gat aga Leu Tyr Aso Arg aag Lys 255 acc tca cac ata Thr Leu His Ile cca Pro 260 cct gat gtt ttc Pro Asp Val Phe aag aaa Lys Lys 265 atg tct gca Met Ser AlIa gac att gtg Asp Ile Val 29S tca Ser 270 caa aag caa tat Gin Lys Gin Tyr t gg T rp 2*75 agt gtt aag agt Ser Vai Lys Ser gaa tat atg Glu Tyr Met 280 ctg tat gag Leu Tyr Giu 987 1035 ctc ttc ttt aaa Leu Phe Phe Lys gtg Val1 290 ggg aaa ttt tat Gly Lys Phe Tyr gag Glu 295 ct~a gat Leu Aso 300 gog gaa tta ggt Aa ISl1u Leu GClv.

cac His 305 aag gag ctt: gac Lvs-C,-s Leu Asp tgg T rp 310 aag atg acc atg Lys Met Thr Met agt Ser 315 ggt gza gga aaa Gly Val Cly Lys tgc Cys 320 aga cag gcz ggt Arg Gin Vai Gly atc Ile 325 tct gaa agt ggg Ser Clii Ser Cly ata Ile 330 1083 1131 1179 gat gag gca gtg Asp Glu Al a Val caa Gin 335 aag cta tta gct Lys Leu Leu Ala cgt Arg 340 gga tat aaa gtt Gly Tyr Lys Val gga cga Cly Arg 345 atc gag cag Ile Clu GI n act ata a-t Thr Ilie Tle 365 cta Leu 350 gaa aca tct gac Ciu Thr Ser Asp caa Gin 355 gca aaa gcc aga Ala Lys Ala Arg ggt. gcc aat.

Gly Aia Asn 360 tca aca gca Ser Thr Ala 1227 1275 cca agg aag cta Pro Arg Lys Leu gtt Val 370 cag gta tta act Gin Val Leu Thr cca Pro 375 agc gag Ser Giu 380 gag atc Glu Ie 395 gga aac atc ggg Gly Asn Ile Gly cct Pro 385 gat gcc gtc cat Asp Ala Val His ctt Leu 390 ctt gct ata aaa Leu Ala Ile Lys 1323 aaa atg gag Lys Met Giu cta caa aag tgt tca act Leu Gin Lys Cys Ser Thr 400 405 gtg tat gga ttt Val Tyr Gly Phe gct Ala 410 1371 ttt gtt gac Phe Val Aso tgt gct gcc ttg agg ttt Cys Ala Ala Leu Arg Phe 415 tgg gtt ggg tcc atc Trp Val Gly Ser Ile agc gat Ser Asp 425 1419 WO 99/19492 WO 9919492PCT/EP98/06977 gat gca tca tgt gct Asp Ala Ser Cys Ala 430 gct. ctt gga gcg tta ttg Ala Leu Gly Ala Leu Leu 435 atg cag gtt tct cca Met Gin Val Ser Pro 440 aag gaa gtg tta tat gac agt Lys Glu Val Leu Tyr Asp Ser 445 aaa Lys 450 ggg cta tca aga Gly Leu Ser Ara gaa Glu 455 gca caa aag Ala Gin Lys 1467 1515 1563 gct cta Ala Leu 460 agg aaa tat acg Arg Lys Tyr Thr t tg Leu 465 aca ggg tct acg Thr Gly Ser Thr gcg Ala 470 gta cag ttg gct Val Gin Leu Ala cca Pro 475 gta cca caa gta Val Pro Gin Val atg Met 480 ggg gat aca gat Gly Asp Thr Asp gct Ala 485 gct gga gtt aga Ala Giy Val Arg aat Asn 490 1611 1659 ata ata gaa tct Ile Ile Glu Ser aac Asn 495 gga tac ttt aaa Gly Tyr Phe Lys ggt Gly 500 tct tct gaa tca Ser Ser Glu Ser tgg aac Trp Asn 505 tgt gct gtt Cvs Ala Val gga gag cta Gly Glu Leu 525 gat Asp 510 ggt cta aa: gaa Gly Leu Asr. Glu t-gt C vs 515 gat gtt gcc ctt Asp Val A'.a Leu agt, gct ct Ser Ala Leu 520 gat gta ct~t Asp Val Leu 1707 1755 att aat cat ctg Ile Asri His Leu tct- Ser 530 agg cta aag cta Arg Leu Lys Leu gaa Glu 535 aag cat Lys His 540 ggg gat att. tEE Gly Asp Ilie Phe Eac caa gtt tac Tyr Gin Val T yr agg Arg 550 ggt tgt ctc aga Gly Cys Leu Arg att Ile 555 gat ggc cag acg Asp Gly Gin Thr atg Met 560 gta aat ctt gag Val Asn Leu Giu ata Ile 565 t: aac aat. agc Phe Asn Asn Ser tgt Cys 570 gtt Val 1803 1851 1899 1947 gat ggt ggt cct Asp Gly Gly Pro tca Ser 575 ggg acc ttg tac Gly Thr Leu Tyr aaa Lys 580 tat ctt gat aac Tyr Leu Asp Asn tgt Cys 585 agt cca act Ser Pro Thr ggt Gly 590 aag cga ctc tta Lys'Arg Leu Leu agg Arg 595 aat tgg atc tgc Asn Trp Ile Cys cat His 600 cca ctc Pro Leu aaa gat gta gaa agc atc aat aaa cgg ctt Lys Asp Val Glu Ser Ile Asn Lys Arg Leu 605 610 acg gca aac tca gaa agt atg caa atc act Thr Ala Asn Ser Glu Ser Met Gin Ilie Thr 620 625 gat gta gtE Asp Val Val 615 gaa gaa ttc Giu Glu Phe 1995 ggc cag tat ctc cac aaa Gly Gin Tyr Leu His Lys 630 2043 WO 99/19492 WO 99/ 9492PCTIEP98/06977 ctt Le u 635 cca gac tta. gaa Pro Asp Leu Giu aga Arg 640 ctg ccc gga cgc Leu Leu Gly Arg atc Ile 645 aag tc agc g-tt Lvs Ser Ser Val cga Arg 650 2091 2139 tca. tca gcc tc Ser Ser Ala Ser gtg Val1 655 ttg cct gct ctt Leu Pro Ala Leu ctg Leu 660 ggg aaa aaa gcg Gly Lys Lys Val ccg aaa Leu Lys 665 caa cga gtt Gin Arg Vai act gat ctg Ile Asp Leu 685 aaa Lys 670 gca ctt ggg caa Ala Phe Gly Gin at c Ile 675 gcg aaa ggg tcc Val Lys Gly Phe aga agt gga.

Arg Ser Gly 680 acg agt tcg Met Ser Leu 2187 2235 ctg ttg gct cca Leu Leu Ala Leu cag Gin 690 aag gaa tca. aac Lys Giu Ser Asn acg Met 695 cct tat Leu Tyr 700 aaa ccc tgt aaa Lys Leu Cys Lys ccc Leu 705 cct aca cca gca Pro Ile Leu Val gga.

Gly 710 aaa agc ggg cca Lys Ser Gly Leu gag Giu 715 cca cc:i ctc tc Leu Ph e Leu Ser caa Gin 720 ccc gaa gca cc Phe Glu Ala Ala aca Ile 725 gac agc gac ticc Asp Ser Asp Phe cca Pro 730 2283 2331 2379 aac tat cag aac Asn Tyr Gin Asn caa Gin 735 gat gtg aca gat Asp Vai Thr Asp gaa Giu 740 aac go: gaa act.

Asn Ala Giu Thr czc aca Leu Thr 745 aca ccc ac Ile Leu lie att cac acc lie His Thr 765 gaa Giu 750 ccc ctt atc gaa Leu Phe Ile Giu aga Arg 755 gca act caa cgg Ala Thr G in Trp tctccrag gc Ser Giu Vai 760 gca acc gca Ala 11ie Ala 2427 2475 aca agc cgc cta Ilie Ser Cys Leu gac Asp 770 gcc ctg aga. cc Val Leu Arg Ser gca agt Ala Ser 780 ccc cct gct gga.

Leu Ser Ala Gly agc Ser 785 acg gcc agg ccc Met Ala Arg Pro ct Val1 790 act cc ccc gaa Ile Phe Pro Giu tca.

Ser 795 gaa gct aca gat Giu Ala Thr Asp c ag Gin 800 aac cag aaa aca Asn Gin Lys Thr aaa.

Lys 805 ggg cca aca ct Gly Pro Ile Leu aaa Lys 810 2523 2571 2619 acc caa gga Ile Gin Gly cct gtt ccg Pro Val Pro cca tgg Leu Trp 815 aat gat Asn Asp 830 cat cca ccc gca His Pro Phe Ala gcc Vai 820 gca gcc gat ggc Ala Aia Asp Gly caa. ttg Gin Leu 825 ata. ccc ccc Ile Leu Leu ggc Gly 835 gag gct aga aga Giu Ala Arg Arg agc agt ggc Ser Ser Gly 840 2667 WO 99/19492 WO 99/ 9492PCT/EP98/06977 agc act cat Ser Ile His 845 cct cgg tca ttg Pro Arg Ser Leu tca Le u 850 ctg acg gga cca Leu Thr Gly Pro aac acg ggc gga Asn Met Gly Gly 855 atc tcc gcc caa Ile Phe Ala Gin 2715 aaa tca Lys Ser 860 act ctt ccc cgt Thr Leu Leu Arg gca Ala 865 aca tgt ctg gcc Thr Cys Leu Ala gt c Val1 870 ctt Leu 875 ggc tgc tc gcg Gly Cys Tyr Val ccg Pro 880 tgt gag tc cgc Cys Glu Ser Cys gaa Glu 885 atc tcc ctc gtg Ile Ser Leu Val gatc Asp 890 2763 2811 2859 act atc ttc aca Thr Ile Phe Thr agg Arg 895 ccc ggc gca cc Leu Gly Ala Ser gat Asp 900 aga atc atg ata.

Arg Ile Met Thr gga. gag Gly Glu 905 agt act ttt Ser Thr Phe gca act cag Ala Thr c-in 925 ttg Leu 910 gca gaa tgc act Vai c-lu Cys Thr gag Glu 915 aca gcg cca gtt Thr Ala Ser Val ccc tag aat Leu Gin Asn 920 aga gga act Arg Gly Thr 2907 295S gat cca cta gta Asp Ser t Leu Val a:c 930 ctt gac gaa ctg Leu Asp Glu Leu ggc Gly 935 agt act Ser Thr 940 ttc gat gga tat Phe Asp Giy Tyr gcc Ala 94S act. gca tct tcg Ile Ala Tyr Ser gt t Val1 950 ccc cgc cat ctg Phe Arg His Leu gca Val1 955 gag aaa gtt caa Giu Lys Val Gin cgc Cys 960 cgg atg ttc ccc Arg Met Leu Phe gca Ala 965 aca cat tat cat Thr His Tyr Hi s cct Pro 970 3003 3051 3099 ccc act aag gaa Leu Thr Lys Glu tcc Phe 975 gcg cc cac cca Ala Ser His Pro cg t Arg 980 gtc acc tcg aaa Val Thr Ser Lys cat atg His Met 985 gct tgc gca.

Ala Cys Ala caa gac cca Gin Asp Leu 1005 agc tc gga Ser Tyr Gly 1020 tcc Phe 990 aaa cca aga tc Lys Ser Arg Ser gat Asp 995 tat caa cca Tyr Gin Pro cgt ggt tgt gat Arg Gly Cys Asp 1000 gtg ttctctg tac cgc Val Phe Leu Tyr Arg 1010 cta act gag gga gcc Leu Thr c-lu Gly Ala 1015 cgc ccc gag Cys Pro c-lu aac caa. gtg Asn Gin Val 3147 3195 3243 cttc caa gtg gca ccc atg gct Leu Gin Val Ala Leu Met Ala 1025 gga ata cca Gly Ile Pro 1030 gct gaa aca gca cca ggt gcc gct caa gcc acg aag aga tca act ggg Val Giu Thr Ala Ser Gly Ala Ala Gin Ala Met Lys Arg Ser Ile Gly 3291 1035 1040 1045 1050 WO 99/19492 28 gga aac ctc aag tca agt gag c:a aga tct. gag ttc tca Glu Asn Phe Lys Ser Ser Giu Leu Arg Ser Giu Phe Ser 1055 1060 gaa gac tgg ccc aag tca ttg grg ggt act tct cga gtc Glu Asp Trp Leu Lys Ser Leu Val Gly lie Ser Arg Val 1070 1075 aat gcc ccc act ggc gaa gat gac tac gac act ttg ttt Asn Ala Pro Ile Gly Giu Asp Asp Tyr Asp Thr Leu PhE 1085 1090 1091 cat gag atc aaa tcc tc tac tgt gtt ccc aaa taaatgc His Glu Ile Lys Ser Ser Tyr Cys Val. Pro Lys 1100 1105 tgacataaca ctatctgaag ctcgttaagt cttttgcctc tctgat aaaaatgctt ar-acatcaaa aaatta:::z czcattaaa aaaaaa.

aaaaaaaa <210> 31.

<211> 1109 <212> PRT <213> Arabidopsis thaliana ecozype Columbia <223> Poiypeptide MSH6 <400> 31 Met Gin Arg Gin Arg Ser Ile Leu Ser Phe Phe Gin Ly 1 5 10 Ala Thr Thr Lys Gly Leu Val Ser Gly Asp Ala Ala Se 25 Gly Ser Gly Gly Pro Arg Phe Asn Val Arg Giu Gly As 40 4 Asp Ala Ser Val Arg Phe Ala Val Ser Lys Ser Val As 55 Gly Thr Asp Thr Pro Pro Giu Lys Val Pro Arg Arg Va 70 75 Gly Phe Lys Pro Ala Giu Ser Ala Gly Asp Ala Ser Se 90 Asn Ile Met His Lys Phe Val Lys Val Asp Asp Arg As 100 105 Glu Arg Ser Arg Glu Asp Val Val Pro Leu Asn Asp Se 115 120 12 PCTIEP98/06977 agt ctg *Ser Leu 1065 gcc cac *Ala His 1080 tgc tta Cys Leu cta cat His aac Asn tgg Trp 3339 3387 3435 3478 aaaa aaaaaaaaaa 3538 3598 3606 5 r p

S

p 1 Pro Thr 1s Gly Gly Ala Lys Glu Val.

Leu Pro Leu Phe Cys Ser 110 Ser Leu Ala Gly Gly Arg Ser Ser Gly Cys WO 99/19492 PCT/EP98/06977 Met Lvs Ala ASn Asp Val Ile Pro Gin Phe I: 135 Thr 145 Arg Thr Asp Lys Val 225 Aso Leu Gin Lys His 305 Arg Leu Ser Leu Pro 385 31n Ser Pro Glu Arg 210 Asn His Tvr Val 290 Lys Gin Leu Asp Val 370 Asr Giu Vai C Gly Met 195 Leu Glu As r Ile Trp 275 Gly Glu Va1 Ala Gin 355 Gin Ala ~rg lu 4et rhr Lys GIy Arg Pro 260 Ser Lys Leu Gly Arc 340 Ala VaJ Va Asn Asp 165 Arg Phe Met Thr Arc; 245 Pro Val Phe Asp Ile 325 Gly Lys L Lei L His His I 150 Ile C Pro Lys Leu Lys 230 Arg Asp Lys Tyr Trp 310 Ser Tyr Ala Thr Leu 390 kla fly krg Glu Gln 215 Phe Pr3 Vai Ser Glu 295 Lys Glu Lys Arg Pro 375 Leu Phe E Val 1 Ala Asp 200 Asp GIu ASo Phe Glu 280 Leu Met Ser Val Gly 360 Ser Ala 3er ksp er L85 Lys Pro Trp Asp Lys 265 Tyr Tyr Thr Gly Gl 345 Ala Th I l Phe Gly 170 Arg Val Val Leu Pro 250 Lys Met Glu Met Ile 330 Arg Asn Ala B Lys krg ;er L55 %sp Leu Pro Cys Glu 235 Leu Met Asp Leu Ser 315 Asp IlE Thi Se3 G1 39! Ser 1 140 Gly I Val Lys Val Gly 220 Ser Ty r Ser Ie Asp 300 Gly Glu Glu Ile Glu 380 .1 Ile ksn krg ?ro Arg Leu 205 Glu Ser Asp Ala Val 285 Ala Val Ala Gin Ile 365 Gly Lys ksn Ala Gly Val 190 Asp Lys Arg Arg Ser 270 Leu Glu Gly Val Leu 350 Pro Asr Met Gly I Glu Pro 175 Leu Ser Lys 7le Lys 255 Gin Phe Leu Lys Gin 335 Glu Arg 1 Ile Glu Lys Leu 160 flu Glu Asn Glu Arg 240 Thr Lys Phe Gly Cys 320 Lys Thr Lys Gly Leu 400 Gin Lys Cys Ser Thr Val Tyr Gly Phe Ala Phe Val Asp Cys Ala Ala 410 415 WO 99/19492 WO 99/ 9492PCTIEP98/06977 Leu Leu Se r Leu 465 Gly Tyr Asn Leu Pro 545 Val1 Thr Leu Asn Met 625 Leu Pro Gly Leu Arg Gly Lys 450 Thr Asp Phe Glu Ser 530 Tvr Asn Leu Leu Lys 610 Gin Leu Ala Gin Gin 690 Phe Ala 435 Gly Gily Thr Lys Cys 515 Arg Gin Leu Tyr Arg 595 Arg Ile Gly Leu Ile 675 Lys T rp 420 Leu Leu Ser Asp Gly 500 Asp Leu Val1 Giu Lys 580 Asn Leu Thr Arg Leu 660 Vai Glu Val1 Leu Ser Thr Aia 485 Ser Val1 Lys Tyr Ile 565 Tyr Trp Asp Giy Ile 645 Gly Lys Ser Giy Met Arg Ala 470 Ala Ser Ala Leu Arg 550 Phe Leu Ile Val1 Gin 630 Lys Lys Giy Asn Ser Gin Giu 455 Vali Giy Giu Leu Giu 535 Gly As n Asp Cys Val1 615 Tyr Ser Lys Phe Met 695 Ile Val 440 Ala Gin Val Ser Ser 520 Asp Cys Asn Asn His 600 Giu Leu Ser Val Arg 680 Met Ser 425 Ser Gin Leu Arg Trp 505 Ala Val1 Leu Ser Cys 585 Pro Giu His Val1 Leu 665 Ser Ser Asp Pro Lys Al a Asn 490 Asn Leu Leu Arg Cys 570 ValI Leu Phe Lys Arg 650 Lys Gly Leu Asp Lys Ala Pro 475 Ile Cys Giy Lys Ile 555 Asp Ser Lys Thr Leu 635 Ser Gin Ile Leu Al a Glu Leu 460 Val Ile Ala G1iu His 540 Asp Gly Pro Asp Ala 620 Pro Ser Arg Asp Tyr 700 Ser Val 445 Arg Pro Giu Val Leu 525 Gly Gly Gly Thr Val1 605 Asn Asp Ala Val Leu 685 Lys Cys 430 Leu Lys Gin Ser Asp 510 Ile Asp Gin Pro Gly 590 Glu Ser Leu Ser Lys 670 Leu Leu Ala Tyr Tyr Val Asn 495 Gly Asn Le Thr Ser 575 Lys Ser Giu Giu Val 655 Aia Leu Cys Al a Asp Thr Met 480 Gly Leu His Phe Met 560 Gly Arg Ile Ser Arg 640 Leu Phe Ala Lys WO 99/19492 WO 99/ 9492PCT/EP98/06977 31 Leu Pro Ile Leu Val Gly Lys Ser Gly Leu Giu Leu Phe Leu Ser Gin 705 710 715 720 Phe Giu Ala Ala Ile Asp Ser Asp Phe Pro Asn Tyr Gin Asn Gin Asp 725 730 735 Val Thr Asp Giu Asn Ala Giu Thr Leu Thr Ile Leu Ile Giu Leu Phe 740 745 750 Ile Giu Arg Ala Thr Gin Trp Ser Glu Val Ile His Thr Ile Ser Cys 755 760 765 Leu Asp Val Leu Arg Ser Phe Al a Ile Ala Ala Ser Leu Ser Ala Gly 770 775 780 Ser Met Ala Arg Pro Val Ile Phe Pro Giu Ser Giu Ala Thr Asp Gin 785 790 795 800 Asn G in Lys Thr Lys Giy Pro il e Leu Lys Ile Gin Giy Leu Trp His 805 810 815 Pro Phe Ala Val Ala Ala Aso G Iy Glin Leu Pro Val Pro Asn Asp Ile 820 825 830 Leu Leu Gly Glu Ala Arg Arg Ser Ser Gly Ser Ile His Pro Arg Ser 835 840 845 Leu Leu Leu Thr Gly Pro Asn Met Gly Gly Lys Ser Thr Leu Leu Arg 850 855 860 Ala Thr Cys Leu Ala Val Ile Phe Ala Gin Leu Gly Cys Tyr Val Pro 865 870 875 880 Cys Giu Ser Cys Giu Ile Ser Leu Val Asp Thr Ile Phe Thr Arg Leu 885 890 895 Gly Ala Ser Asp Arg Ile Met Thr Gly Giu Ser Thr Phe Leu Val Giu 900 905 910 Cys Thr Glu Thr Ala Ser Val Leu Gin Asn Ala Thr Gin Asp Ser Leu 915 920 925 Val Ile Leu Asp Glu Leu Gly Arg Gly Thr Ser Thr Phe Asp Gly Tyr 930 935 940 Ala Ile Ala Tyr Ser Val Phe Arg His Leu Val Glu Lys Val Gin Cys 945 950 955 960 Arg Met Leu Phe Ala Thr His Tyr His Pro Leu Thr Lys Glu Phe Ala 965 970 975 Ser His Pro Arg Val Thr Ser Lys His Met Ala Cys Ala Phe Lys Ser 980 985 990 WO 99/19492 WO 99/ 9492PCT/EP98/06977 32 Arg Ser Asp Tyr Gin Pro Arg Gly Cys Asp Gin Asp Leu Val Phe Leu 995 1000 1005 Tyr Arg Leu Thr Giu Gly Ala Cys Pro Giu Ser Tyr Gly Leu Gin Val 1010 1015 1020 Ala Leu Met Aia Gly Ile Pro Asn Gin Vai Val Giu Thr Ala Ser Gly 1025 1030 1035 1040 Ala Ala Gin Ala Met Lys Arg Ser Ile Gly Giu Asn Phe Lys Ser Ser 1045 1050 1055 Giu Leu Arg Ser Glu Phe Ser Ser Leu His Giu Asp Trp Leu. Lys Ser 1060 1065 10"70 Leu Val Gly Ile Ser Arg Val Ala His Asn Asn Ala Pro Ile Gly Giu 1075 1080 1085 Asp Asp Tyr Asp Thr Leu Phe L eu Trp His Giu Ile Lys Ser Ser 1090 1095 1100 Tyr Cvs Val Pro Lys 1105 .<210> 32 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Forward primer for PCR amplification of ATHGENEA microsatellite <400> 32 accatgcata gcttaaactt cttq 24 <210> 33 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for PCR amplification of ATHGENEA microsatellite <400> 33 acataaccac aaataggggt gc 22 WO 99/19492 WO 9919492PCT/EP98/06977 34 <211> 18 <212> DNA <213.> Artificial sequence <220> <223> Forward primer DMCIN-A for PCR on genomic DNA of Arabidopsis thaliana ssp. Landsberg erecta "Ler" <400> 34 gaagcgatat tgttcgtg 18 <210> <211> 18 <212> DNA <213> Artificial sequence <220> <223> Reverse primer DMCIN-B for 2CR on genomic DNA of Arabidopsis thaliana ssp. Landsberg erecta 'Ler" <400> agattacgag aacaztcc 18 <210> 36 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Forward primer DMCIN-1 for 2CR on genomic DNA of Arabidopsis thaliana ssp. Landsberg erect~a "Ler" <400> 36 acgcgtcgac tcagctatga gattac-tcgt g 31 <210> 37 <211> 29 <212> DNA <213> Artificial sequence <220> <223> <400> Reverse primer DMCIN-2 for PCR on genomic DNA of Arabidopsis thaliana ssp. Landsberg erecta. "Ler" 37 gctctagatt tctcgctcta agactctct WO 99/19492 WO 9919492PCT/EP98/06977 34 <210 38 <-211> 32

DNA

<213> Artificial sequence <220> <223> Forward primer DMCIN-3 for 2CR on genomic DNA of Arabidopsis thaliana ssp. Landsberg erecta "Ler" <400> 38 gctctaqagc ttctcttaag taagtgattg at 32 <210> 39 <211> 48 <212> DNA <213> Artificial seauence <220> <.223> Reverse primer DMCIN-4 for 2CR on genomic DNA of Arabidopsis t-haliana ssp. Landsberg erecta "Ler' <400> 39 tcccccgggc tcgagagatc tccazqggttt cttcagctct atcaatcc 48 <210> <211> 26 <212> DNA <213> Artificial sequence <220> <223> Forward primer DMC~a for 2CR on genomic DNA of Arabidopsis thaliana ssp. Landsberg erecta 'Ler" <400> acgcgtcgac gaattcgcaa gtgggg 26 <210> 41 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Reverse primer DMC~b for 2CR on geflomic DNA of Arabidopsis thaliana ssp. Landsberg erecta "Ler" <400> 41 WO 99/19492 WO 9919492PCTIEP98/06977 tccatggaga tctcccgggt accgatttgc ttcgaggg <210 <211> <212> <213> <220> <223> <400> 42

DNA

Artificial sequence Forward primer for PCR amplification of ATEATi SSLP marker in Arabidopsis thaliana subspecies 42 gccactgcgt gaatgatatg <210> <211> <212> <213> 43 22

DNA

Artificial seauence <220> <223> Reverse primer for PCR amplification of ATEAT1 SSLP marker in Arabidopsis thaliana subspecies <400> 43 cgaacagcca acattaacttc cc 22 <210> 44 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Forward primer for PCR amplification of NGA63 SSLP marker in Arabidopsis thaliana subspecies <400> 44 aaccaaggca cagaagcg 1~8 <210> <211> 18 <212> DNA <213> Artificial sequence <220> 223>Reverse primer for PCR amplification Of NGA63 SSLP marker in Arabidopsis thaliana Subspecies WO 99/1 9492 PTE9/67 PCT/EP98/06977 <400> acccaagtga tcgccacc 1 <210> 46 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Forward primer for PCR amiolification of NGA248 SSLP marker in Arabidopsis thaliana subspecies <400> 46 taccgaacca aaacacaaag g <210> <211> <212> <213> <220> <223> <400> 47 22

DNA

Artificial sequence Reverse primer for 2CR amplification of NGA248 SSLP marker in Arabidopsis thaliana subspecies 47 cctgtatctc ggrtgaactct cc <210> <211> <212> <213> <220> <223> <400> 48 22

DNA

Artificial sequence Forward primer for PCR amnolification of NGA128 SSLP marker in Arabidopsis thaliana subspecies 48 ggtctgttga tgtcgtaagt cg <210> <211> <212> <213 <220> 49 22

DN'A

Artificial sequence WO 99/1 9492 PTE9/67 PCT/EP98/06977 <223> <400> Reverse primer for PCR amplification of NGA128 SSLP marker in Arabidopsis thaliana subspecies 49 atcttgaaac cr-ttagggag gg <210> <211> <212> <213> 22

DNA

Artificial sequence <220> <223> <400> Forward primer for 2CR amplification of NGA280 SSLP marker in Arabidopsis thaliana subspecies so ctgatctcac ggacaatagt gc <210> <211> <212> <213> <220> <223> <400> 51

DNA

Artificial secruence Reverse primer for 2CR amplification of NGA280 SSLP marker in Arabidopsis thaliana subspecies 51 ggctccataa aaagtgcacc <210> 52 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Forward primer for PCR amplification of NGA111 SSLP marker in Arabidopsis thaliana subspecies <400> 52 ctccagttgg aagctaaagg g 21 <210> 53 <211> 21 <212> DNA <213> Artificial sequence WO 99/19492 PCT/EP98/06977 '-220> ':Z2 3 <400> 38 Reverse primer for 2CR amplification of NGA111 SSLP marker in Arabidopsis thaliana subspecies 53 tgttttttag gacaaatggc g <210> <211> <212> <213> <220> <223> <400> 54

DNA

Artificial sequence Forward primer for PCR amplification of NGA168 SSLP marker in Arabidopsis thaliana subspecies 54 ccttcacar-c caaaacccac <210> <211> <212> <213> <220> <22]> <400>

DNA

Artificial sequence Reverse primer for PCR amplification ofi NGA168 SSLP marker in Arabidopsis thaliana subspecies gcacataccc acaaccagaa <210> <211> <212> <213> <220> <223> <400> 56

DNA

Artificial sequence Forward primer for 2CR amplification of NGAl126 SSLP marker in Arabidopsis thaliana subspecies 56 cgctacgctt ttc9-gtaaag WO 99/19492 PCTIEP98/06977 39 <210> 57 <211> <212> DNA <213> Artificial sequence <220> <223> Reverse primer for PCR amplification of NGA1126 SSLP marker in Arabidopsis thaliana subspecies <400> 57 gcacagtcca agtcacaacc <210> <211> <212> <213> <220> 2 23> <400> 58

DNA

Artificial sequence Forward primer for PCP. amplification of NGA361 SSLP marker in Arabidopsis r-haliana subspecies 58 aaagagatga gaatttggac <210> <211> <212> <213> <220> <223> <400> 59 23

DNA

Artificial sequence Reverse primer for PCR amplification of NGA361 SSLP marker in Arabidopsis thaliana subspecies 59 acatatcaat atattaaagt agc <210> <211> <212> <213> <220> <223> 18

DNA

Artificial sequence Forward primer for PCR amplification of NGA168 SSLP marker in Arabidopsis thaliana subspecies <400> WO 99/19492 PCT/EP98/06977 tcgtcaccg cactgccg 1 <210> 61 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for PCR amplification of NGAl6B SSLP marker in Arabidopsis chaliana subspecies <400> 61 gaggacatgt ataggagcct cg 22 <210> 62 <211> <212> DNA <213> Artificial seauence <220> <223> Forward primer "or PCR amplificacion of AthB702 SSLP marker in Arabidonsis thaliana. subspecies <400> 62 tgacccccc ttccatggag <210> 63 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for PCR amplification of AthBIO2 SSLP marker in Arabidopsis thaliana subspecies <400> 63 ttaacagaaa cccaaagctt tc 22 <210> 64 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Forward primer for PCR amplification of AthUBIQUE SSLP marker in Arabidopsis thaliana subspecies WO 99/19492 WO 99/ 9492PCT/EP98/06977 <400> aggcaaatgt ccatttcatt g <210> <211> <212> <213> <220> <223> <400>

DNA

Artificial seauence Reverse primer for PCR amplification of AthUBIQUE SSLP marker in Arabidopsis thfaliana subspecies acgacacggc agatttctcc <210> <211> <212> <213> <220> <223> <400> 66 21

DNA

Artif.~cial seqo-ence Forward primer for PCP. amplification of NGA172 SSLP marker in Arabidopsis thaliana subspecies 66 agctgcttcc ttatagcgtc c <210> <211> <212> <213> <220> <223> <400> 67 19

DNA

Artificial sequence catccgaatg cc Reverse primer for PCR amplification of NGA172 SSLP marker in Arabidopsis thaliana subspecies 67 :at tgt tc 19 68 21

DNA

Artificial sequence <210> <211> <212> <213> <220> WO 99/19492 PCT/EP98/06977 42 Forward primer for PCR amplificazion of NGA126 SSLP marker in Arabidopsis thaliana subspecies .400> 63 gaaaaaacgc tactttcgtg g 21 ':210> 69 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for 2CR amplification of NGA126 SSLP marker in Arabidopsis thaliana subspecies <400> 69 caagagcaat atcaagaaca gc 22 <210> <211> <212> <213> <220> <223> <400>

DNA

Artificial sequence Forward primer for PCR amplification of NGA162 SSLP marker in Arabidopsis thaliana subspecies catgcaattt gcatctgagg <210> <211> <212> <213> <220> <223> <400> 71 22

DNA

Artificial sequence Reverse primer for 2CR amplification of NGA162 SSIJP marker in Arabidopsis thaliana subspecies 71 ctctgtcact cttttcctc gg <210> <211> <212> <213 72 21

DNA

Artificial sequence WO 99/19492 PCTIEP98/06977 43 <:220> <1223 Forward primer for PCR amplification of NGA6 SSLP marker in Arabidopsis thaliana subspecies ':400> 72 tggatttctt cctctcttca c 21 <210> 73 <211> 21 ':212> DNA ':213> Artificial sequence <:220> <:223> Reverse primer for PCR amplification of NGAG SSLP marker in Arabidopsis thaliana subspecies ':400> 73 acggagaaac ttacactgat c 21 ':210> 74 ':211> <212> DNA ':213> Artificial sequence <:220> <:223> Forward primer for 2CR amplification of NGA12 SSLP marker in Arabidopsis thaliana subspecies ':400> 74 aatgttgtcc tcccctcctc <210> ':211> 22 ':212> DNA <:213> Artificial sequence <:220> <:223> '400> Reverse primer for PCR amplification of NGA12 SSLP marker in Arabidopsis thaliana subspecies 7 tgatgctctc tgaaacaaga gc WO 99/19492 PCT/EP98/06977 44 <210> 76 <211> 21 <212>

DNA

<213> Artificial sequence <220> <223> Forward primer for PCR amplification of NGA8 SSLP marker in Arabidopsis thaliana subspecies <400> 76 gagggcaaat cttctatttcg g 22' <210> 77 <211> 22 <212>

DNA

<213> Artificial sequence <220> <223> Reverse primer f'cr PCR am:Dlification of NGA8 SSLP marker in Arabidousis zha 1 lana subsoecies <400> 77 tggctttcgt ttataaacat cc 2 <210> 78 <211> 21 <212>

DNA

<213> Artificial sequence <220> <223> Forward primer for PCR amplification of NGA1107 SSLP marker in Arabidopsis thaliana. subspecies <400> 78 gcgaaaaaac aaaaaaatcc a 22 <210> 79 <211> 21 <212>

DNA

<213> Artificial sequence <220> <223> Reverse primer for PCR amplification of NGA1107 SSLP marker in Arabidopsis thaliana subspecies <400> 79 WO 99/19492 WO 9919492PCT/EP98/06977 cgacgaaccg acagaattag g <210> <211> <212> <213> <220> <223> <400> 21

DNA

Artificial sequence Forward primer for PCR amplification of NGA225 SSLP marker in Arabidopsis thaliana subspecies gaaatccaaa tcccagagag g <210> <211> <212> <213> <220> <223> <400> 81 22

DNA

Artificial sequence Reverse primer for PCR amplificauion of NGA225 SSLP marker in Arabidopsis thaliana subspecies 81.

tctccccact agttttgtgt cc <210> 82 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Forward primer for PCR amplification of NGA249 SSLP marker in Arabidopsis thaliana subspecies <400> 82 taccgtcaat ttcatcgcc 19 <210> <211> <212> <213> <220> <223> 83 22

DNA

Artificial sequence Reverse primer for PCR amplification of NGA249 SSLP marker in Arabidopsis thaliana subspecies WO 99/19492 WO 99/ 9492PCT/EP98/06977 <400> 83 ggatccctaa ctgtaaaatc cc <210> <211> <212> <213> <220> <2 23 <400> 84 22

DNA

Artificial sequence Forward primer for PCR amplification of CA72 SSLP marker in Arabidopsis thaliana subspecies 84 aatcccagta accaaacaca ca 2 <210> <211> <212> DNA <213kArtific--al secruence <220> <223> Reverse primer for PCR amplification of CA72 SSLP marker in Arabidopsis thaliana subspecies <400> cccagcctaa ccacgaccac <210> <211> <212> <213> <220> <223> <400> 86

DNA

Artificial sequence Forward primer for PCR amplification of NGA151 SSLP marker in Arabidopsis thaliana subspecies 86 gttttgggaa. gttttgctgg <210> <211> <212> <213> <220> 87 24

DNA

Artificial sequence WO 99/19492 PCT/EP98/06977 47 <223> Reverse primer for PCR amplification of NGA151 SSLP marker in Arabidopsis thaliana subspecies <400> 87 cagtctaaaa gcgagagtat gatg 24 <210> <211> <212> <213> <220> <223> <400> 88 22

DNA

Artificial sequence Forward primer for PCR amplification of NGA106 SSLP marker in Arabidopsis thaliana subspecies 88 gttatggagt tctagggca cg <210> <211> <212> <213> <220> <223> <400> 89

DNA

Artificial sequence Reverse primer for PCR amplification of NGA106 SSLP marker in Arabidopsis thaliana subspecies 89 tgccccattzl gttcttctc <210> <211> <212> <213> <220> <223> <400>

DNA

Artificial sequence Forward primer for PCR amplification of NGA139 SSLP marker in Arabidopsis thaliana subspecies agagctacca gatccgatgg <210> 91 <211> 21 <212> DNA <213> Artificial sequence WO 99/19492 PCT/EP98/06977 <220> <223> <400> 48 Reverse primer for PCR amplification of NGA139 SSLP marker in Arabidopsis thaliana subspecies 91 ggtttcgttt cactatccag g <210> <211> <212> <213> <220> <223> <400> 92 22

DNA

Artificial sequence Forward primer for PCR amplification of NGA76 SSLP marker in Arabidopsis thaliana subspecies 92 ggagaaaatg tcaccctcca cc 22 <210> 93 <211> <212> DNA <213> Artificial sequence <220> <223> Reverse primer for PCR amplification of NGA76 SSLP marker in Arabidopsis thaliana subspecies <400> 93 aggcatggga gacatttacg <210> 94 <211> <212> DNA <213> Artificial sequence <220> <223> <400> Forward primer for PCR amplification of ATHS0191 SSLP marker in Arabidopsis thaliana subspecies 94 ctccaccaat catgcaaatg WO 99/19492 WO 9919492PCT/EP98/06977 49 <210> <211> 21 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for PCR amplification of ATHS0191 SSLP marker in Arabidopsis thaliana subspecies <400> tgatgttgat ggagatggtc a 21 <210> <211> <212> <213> 96 22

DNA

Artificial sequence <220> <223> Forward primer for PCR amplification of NGA129 SSLP marker in Arabidopsis thaliana subspecies <400> 96 tcaggaggaa ctaaagtgag gg 22 <210> 97 <211> 22 <212> DNA <213> Artificial sequence <220> <223> <400> Reverse primer for PCR amplification of NGA129 SSLP marker in Arabidopsis thaliana subspecies 97 cacactgaag atggtcttga gg <210> <211> <212> <213> <220> <223> <400> 98 8062

DNA

Arabidopsis thaliana ecotype Columbia Genomic DNA sequence of AtMSH6 97 ttttttggtt gctaacaata aaggtatacg gttttatgtc atcaatataa ctatatataa WO 99/19492 WO 9919492PCT/EP98/06977 aagaaatgaa ttacttttta ttttacccct gaacagcttt ccaaatccgc ctgctttcac tcaccggaac tttgaaccgc ggctcttttg ggtatctgct tctgagtt tttatatata ctggaacgt t ctttttgggt aatttggttt taaaatttat ggggttCagc tatacaataa aaaaaaagt t ttttggcgcc cactctctct aaacccacgg ggcagcggag gttttgctgt ttccgcgtcg ccctgttctc agaggtacta agatatatat cat tggtcaa aagtttaaca accattctca rttgtgacatc acgatccac cgccgtagat gacaccgttt t t C gattCtg cggcatcgtc gaaattgaga cgaagat tgg cttagagatg tggattcgtg tcggttcggt ttcggtttca gaaccgaata tagaacataa gagccctgag aatctttccc cacaattcca cggcgactac accacgattt ttcgaaatct tgtcctgccg caatattatg atcttcgatt tgttttttca caaaatacaa cctagaacct tattcctgaa ttcttctcca gccgcaggct ttcccctttt ctcagagctg tggagaacta gatgaat tga aacgar-gaag t ttctaggaga attgacgacg gtacaccata tatgagt tat attcggatcc ccaatgcctg atcggacggt gagtatcgctt ccctttcgaa aaaaatgcag gaagggtttg aatgtgaagg gtcgatgagg tctggattta cataagtttg ctcttaattt C ttatcaaac gataaacgac tctccatctt ctacctgagt atctcgctct ctgtttcttc tgttcgaacc cgt taaccgc c tggtCcc ca gagaaaggaa atgaaaaacg atrcgatcat tgtcattatc ttacccgatt cat taaaa tt cttacttcca attgactcgt catcaaagcc tccgccattt ttctctcagc cgccagagat gtttccggcg aaggggatgc ttagaggaac agccggctga C aaaagt cga tgttatcttt aaaacaacaa a--cgtttaac c gc aag ca ca cctctcattg ctgtatcatc ttccagcttc ggcaccgaat tztcggatcg gtccttgtgtt caacgcgaaa t:crtgagag Ct:Zzttctcc tgar.ttgcag :ggctcaatg aaactaacca gaaccgaatt Z:ggctagaaa tcaaagagtg ctacgacgca tcaaaacatc cgattttgtc atgctgctag taaaggcgac ggat actc ca atccgccggt tgatcgagat agctggaaga gacttttttt catttcccaa gcc tgat tag at ct gt tCcg aacctcacct ttcgtgttaa ttcttaaccg cgtaggtctt actgctcctg attttattaa gattgtgata atcttcgtct ttaaccaatg gttttatata aaaattttcg attcgaaacc gatccaacgg aacagtcaac aggcgaaaat gtttctctct tttcttccaa cggcgggggc gcttctgtac c cggagaagg gatgcttcgt tgttctggag agaagattcg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 WO 99/1 9492 PTE9/67 PCT/EP98/06977 tgtaatttgt ttttcaggag atgatgttat ttagtttcag ttcctggtcc aggatgaaat aaatgctcca aatggcttga acgatagaaa agcaatattg gtaactatta ttacgttatg gt cacaagga attagttgaa tgtttcaggt gtgggtaagg aagaaatgga ttattcgtta tgtgtgctca agt tacggat tt t caagc c gattcatatg agatataaag gctaatactg tttctttttc tatcagataa aacatcgggc cgtattcgtt ggagagattc tgattactgc at.Cggatcgt tgtttacaaa ccgagaagat gttgttccgc tcctcaatct tgggagagct agaaacacca gacttttaag ggatccggtt gtcttctcga gaccttacac gagtgttaag atctagtgt ctcttctcag gct tgactgg acaactggcc tggtatctct gaaccatcat aatcacttca tataataagt ctggcaaggc aatatatgtt tgtgttgaaa ctttcctatt t tggacgaat taagttttct tgtacattac CC ccaaggaa ctgatgccgt cgtcecaata gaacttagat gggatgcgt c gaggataagg tgtggagaga atcagggatg ataccacctg agtgaatata caatccattt gggaaatct aagatgacca CgcCtgaaCC gaaagtggga actttatgga agcatcatta ctCCtcatca atatataccc tacttgtatg ctggatagt t ctactaatta cgagcagctCa Cggataggtc tttcatgctg gctagt ecag ccatcttctt tgaacgattc atggtaaaac cagtagaaga cacgtgct tc ttcctgtatt agaaagaagt ccaatagaag atgttttcaa tggacatCgt ccrtcaatgtg atgagctgta C gagtggtgt attgtgtcta C agatgaggc attcgtCtac gttaggatcc aggagt aaca agctaacctt tggtaccctc acatatgctt atctctatgC C caagaaaga tataggagta tcgcttgaag ggactctaac aaacgaagga acgtcctgat gaaaatgtct gctttcttC atttgCtcac tgagctagat gggaaaatgc taaattttga agtgcaaaag tgctacttcg Cgagaactca aacaaaact t tgctagttca attttgtctc ccaacagaaa atgaaggcta aaccatgct C gatggcgatg cgagttctgg aaaaggctga accaaatttg gatccccttt gcatcacaaa aaagtggtta ttacatctgt gcggaattag agacaggt aa caccaccttt ctattagctc gctaggattC ggatgCCCtc gcacaatatt ctgtagtaac tcatggaggc ctagcatgca tcCCCCCgaa agccagaggt tCttgatca ctggtctgat aagcgaggga tttacttatt 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 tgtattgaca cactcgttgt gaaacatctg accaagcaaa aaggagagtg ttgcagactg CaaCtaactc aatggctatt gtattaactc catcaacagc gctataaaag aggtttgtta WO 99/19492 WO 99/ 9492PCT/EP98/06977 52 tacctatca tgttcagttc atccaagtcc tgaaaaatta cactcttctt taccaatctt ccatcaagct gtgtaaagga tttggaatta tgcaagaggt ttttaagtga tatcattcct tctcgtgtgc tgct caggaa C cttt at t t gttgactgtg gccctggag tgacttcctg ttttccagg ccagttgttt atgttgaaat gacaggtacc tctcttaaac gtacagttgg ataatagaat ggtccaaatg aggctaaagg ttcataactt ttttccatac tgagatattt ataacttcaa agtaagtaat tcccttgaaa agatctgtrtt atcccctact ttcttacaat tgttactgca tccccctctt tggagaaaat gatagctgat ttgcctttgg ttacattgcc agatcaaaat ggagctacaa ctgccttgag gttttgggtt cgttattgat gcaggtaagc tgcatatatt tgggttgcag tttctccaaa ggaagtg~za tgttaaacag aatttaaggt attgcagggc tatcaagaga atttcagtag gcaagctaac attgctaatg tagatgatgt ctccagtacc acaagtaatg ctaacggata ctttaaaggt aatgtgatgt tgcccttagt tgtgttggct tgtttagct acagtttcta tctacttgca caagtttaca ggggttgtc.

aacaatagct gtgatggtgg ctagagggca gacatagaag agccgatttt tgtttaccta gaaaatcatt atttgatgct aagattcttt gcacttgaaa gatcatagtc tgcaattgca tcttaatgac cattgtctat ccaagctgta cattatcatg taatggtttg atgtcaattt aagtgtZcaa ctgtgtatgg gggtccatca gcgatgatgc aagtgtattc tgtatcttat gaactaattc tgaatcacca tatgracagta aaggtaaact aaatgacact ggttaattta agcacaaaag gctctaagga tgacaaCta accgctcacc ttgttttata aattcaatct tgtcaagtaa gttgtgtttg attaagtagc ttcttctgaa atttgctttt atcatgtgct gtgtaccatg tttggtatgt gcttgtatcg aagtgcatac aatatacgrt gaatgatagg gtctacggcg agttagaaat tgctgttgat tcatctgtct aaaggaactt atggggatat tggt aaat ct ttcttttttg acggatctcc atcttgataa cactcaaaga 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 ttatgtttca ggggatacag tcttctgaat gctcttggag ttgcttttca gctagaagat cagaattgat tccttcaggc gaaaaattct tgtagggacc aaggaat tgg atctaatagg atgctgctgg catggaactg agctaattaa caaattaagc gtacttaagc ggccagacga aagtgcatat aatacttcgt ttgtacaaat atctgccatc ctgtgttagt ccaactggta agcgactctt tgtagaaagc atcaataaac ggcttgatgt agttgaagaa ttcacggcaa actcagaaag WO 99/19492 WO 99/ 9492PCT/EP98/06977 tatgcaaatc catcaagtct gctgaaacaa ataggactaa gaaagggttc gagtttgctt atttctttct ctatctttca gatctcaact gatggtttga agatgaaaac atggtctgag agcaagtctc agatcagaat tgcagttgca aagaagcagt aaaatcaact cgttagataa ttctgtctgc actatcttca tctcaaaata tttgcttttt atgcaactca atggatacgc caacaaaaac actggccagt atctccacaa agcgttcgat catcagcctc cgagtaagta tcaatcacaa aacattacgg gtctagctaa catttctctt aacttattgc agaagtggaa ttgatctgtt tataaactct gtaaacttcc caattcgaag cagccataga tactttacaa caaaatgtct cacacccga ggatcctaaa tttatttcta acttgaactt gctgaaactc gtcattcaca t ctgctggaa cagaaaacaa gccgatgc ggcagcatc cttcttcgtg ttactctatt agcttggctg caaggct tgg ccaattcctc tttzaggtacc ggat tcacta cattgcatac atgcaattca tcacaatact ccataagctg gcatggccag aagggccaat aattgcctgt atcctcggtc caacatgtct ctttgcaatc ctacgtgccg cgcatctgat gaactattta tttttggtag gtaatccttg tcggtaacct ttttgctgaa acttccagac ttagaaagac tgtgttgcct gctcttctgg g'ttttctgag taatgccttc agactgttct ccttcttttg attgcaggtt aaagcatttg gttggctcta cagaaggaat tatattagta ggaaaaagcg tagcgacttt ccaaattatc gtcactactc aaagcaatgc gggatttgct t-tt~attcct attaatcttg taccagaacc tatcgaactt tttatcgaaa cctagatgtc ctgagatct gcctgttatt tttcccgaat acttaaaatc caaggactat tccgaatgat atactccttg attgttactg acgggaccaa ggccgttatc tttgcccaag agttcttcaa catgaataat tgtgagtctt gcgaaatctc agaatcatga caggagagag ctcagatttt gtctgattgg aatgcactga gacagcgtca acgaactggg cagaggaact gctcttctcc ttcaacttat acttattgat ttatatcagg tgc tcggacg ggaaaaaagc cacgagtagt caatgtctgg ggcaaac tgt caaatatgat ggctagagt t aggtgcccat atatggctta aatgtttttg aagatgtgac gagcaactca ttgcaatcgc cagaagctac ggcatccat gcgaggctag acatgggcgg tttgtatact aaattctgtt cctcgtggat taagttttgt acaaggtggt gt tttcaga agtactttcg acttgttgat tttttcgtca 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 cctggtagag aaagttcaat gtcggatgct Ctgac atcaccccca ctttgcaaca cattaccacc ctctcaccaa WO 99/1 9492 PTE9/67 PCT/EP98/06977 54 ggaattcgcg tctcacccac gtgtcacctc gaaacacatg gcttgcgcat tcaaatcaag atctgattac caaccacgtg gggagcttgt cctgagagct agtggttgaa acagcatcag caagtcaagt gagctaagat ggtgggtatt tctcgagtcg gacataacac tatctgaagc aaaatgctta tatatcaaaa tcggtttagc tatggtatat acaaacagta tattaagaag gcaaataacc tcgactaaag aagttctgaa ttccctaaaa caacacaaac ctacatagtt tccatttctt catcttcact tcacgtaacc tagcaatctc aaatctctct gaaaatcagc ttctccatct tctcataaac gaagcagcgt cattaaccct tcttcagaat caccaggact accttagtgt tcataaacac gacttgggcg aaacacaaag cctaaacctg atttctcaac atcattctcc tacattgcgt gctacttcat tccaatcctc ccccaaactt caagcaacac gctgtgatca acggacttca gtgctgctca ctgagttctc cccacaacaa agatcaaatc tcgttaagtc aattgtttcc aatatatgta gaaatatgt t t tgcaaagac c caaaaaaaa tataacttac ctcatcatca actctgagct tctcatcttc cttcccaaac cttcctctcg atccatcatc aaactcacct caacatatcc cttaatcttc c tcagat t c gttcctcaaa aa agacctagtg agtggcactc agccatgaag aagtctgcat tgcccccatt ctcttactgt ttttgcttct tcgattataa tgttcatgag tatgcattaa caaacacaaa aaacagaaca tcatcactga tcaccaccac ctatcaacaa tccaactcct ctctcaacag tactcaacct tcatcaaacc gaat caacac aacttattga tcttttctat tccatcctta Ctccttctac ttcttgtacc atggctggaa agatcaattg gaagac tggc gqcgaagatg gttcccaaat ctgatgttta caagattata attggtcaag tttaagtttc ttacaaaact tattttgttg gattaacatc catgatgatt tctgcttctC tcattgctc aatccgccaa catcatcctc cattagactt cacaagctaa aaaacgacca acttcctctt gctcctcact ccaattgcaa gt Ctaaccga taccaaacca gggaaaact t tcaagtcatt actacgacac aaatggctat ttcctcttaa tatgtatctg agaaatactc aagataaact tataagactt catctacaaa agaatcattc ctcctcctct ctgcaactcc tttcttactc catcttatac atcctcctcc atctaaataa acctaaatCC tttacttgaa caagtcatca cactttctca aaacctatct 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8062

Claims (125)

1. An isolated and purified DNA polynucleotide comprising a nucleotide sequence encoding a plant polypeptide functionally involved in the DNA mismatch repair system of said plant, wherein said polypeptide is homologous to AtMSH3 (SEQ ID NO: 19) or to AtMSH6 (SEQ ID NO: 31).

2. A DNA polynucleotide according to claim 1, wherein said plant is a higher plant.

3. A DNA polynucleotide according to claim 2, wherein said plant is selected from members of the Brassicaceae, the Poaceae, the Solanaceae, the Asteraceae, the Malvaceae, the Fabaceae, the Linaceae, the Canabinaceae, the Dauaceae, and the Cucurbitaceae.

4. A DNA polynucleotide according to claim 2, wherein said plant is selected from maize, wheat, oats, barley, rice, tomato, potato, tobacco, capsicum, sunflower, lettuce, artichoke, safflower, cotton, okra, beans, peas, melon, squash, cucumber, oilseed rape, 15 broccoli, cauliflower, cabbage, flax, hemp, hops and carrot.

5. A DNA polynucleotide according to claim 2, wherein said plant is Arabidopsis thaliana.

6. A DNA polynucleotide according to any one of claims 1 to 5, wherein said polypeptide is homologous to a mismatch repair polypeptide of a yeast or of a human.

7. A DNA polynucleotide according to claim 1, wherein said nucleotide sequence is selected from the polypeptide-encoding portions of gene AtMSH3 (SEQ ID NO: 18) and gene AtMSH6 (SEQ ID

8. A DNA polynucleotide according to claim 1, comprising a nucleotide sequence selected from nucleotides 100 to 3342 of SEQ ID NO:18, or nucleotides 142 to 3468 of 25 SEQ ID

9. A DNA polynucleotide according to claim 1 wherein said polypeptide is AtMSH3 (SEQ ID NO: 19). A DNA polynucleotide according to claim 1 wherein said polypeptide is AtMSH6 (SEQ ID NO: 31).

11. An isolated and purified DNA polynucleotide comprising a nucleotide sequence encoding a DNA mismatch repair protein active in plants, or encoding a fragment of said protein having the biological activity thereof, and wherein said protein or fragment thereof shares at least 50% identity with the amino acid sequence of AtMSH3 (SEQ ID NO: 19) or AtMSH6 (SEQ ID NO: 31).

12. An isolated and purified DNA polynucleotide comprising a nucleotide sequence encoding a DNA mismatch repair protein active in plants, or encoding a fragment of said protein having the biological activity thereof, and wherein said nucleotide sequence A395498auspcci 31 shares at least 50% identity with the nucleotide sequence of AtMSH3 (SEQ ID NO: 18) or AtMSH6 (SEQ ID NO:

13. An isolated and purified DNA polynucleotide comprising a nucleotide sequence encoding a DNA mismatch repair protein active in plants, or encoding a fragment of said protein having the biological activity thereof, substantially as hereinbefore described with reference to any one of the examples, but excluding reference to any non- plant sequences.

14. An isolated and purified plant polypeptide functionally involved in the DNA mismatch repair system of said plant, wherein said polypeptide is homologous to AtMSH3 (SEQ ID NO: 19) or to AtMSH6 (SEQ ID NO: 31). A polypeptide according to claim 14, wherein said plant is a higher plant.

16. A polypeptide according to claim 15, wherein said plant is selected from members of the Brassicaceae, the Poaceae, the Solanaceae, the Asteraceae, the Malvaceae, the Fabaceae, the Linaceae, the Canabinaceae, the Dauaceae, and the Cucurbitaceae.

17. A polypeptide according to claim 15, wherein said plant is selected from maize, wheat, oats, barley, rice, tomato, potato, tobacco, capsicum, sunflower, lettuce, artichoke, safflower, cotton, okra, beans, peas, melon, squash, cucumber, oilseed rape, broccoli, cauliflower, cabbage, flax, hemp, hops and carrot.

18. A polypeptide according to claim 15, wherein said plant is Arabidopsis thaliana.

19. A polypeptide according to any one of claims 14 to 18 which is homologous to a mismatch repair polypeptide of a yeast or of a human. A polypeptide according to claim 14, wherein said polypeptide is AtMSH3 25 (SEQ ID NO: 19) or AtMSH6 (SEQ ID NO: 31).

21. An isolated and purified plant polypeptide functionally involved in the DNA mismatch repair system of said plant, selected from the group consisting of a polypeptide encoded by the gene AtMSH3 (SEQ ID NO: 18), a polypeptide encoded by the gene AtMSH6 (SEQ ID NO:30), polypeptides homologous to a polypeptide encoded by the gene AtMSH3 (SEQ ID NO: 18) and polypeptides homologous to a polypeptide encoded by the gene AtMSH6 (SEQ ID

22. An isolated and purified polypeptide encoded by the gene AtMSH3 (SEQ ID NO: 18) or by the gene AtMSH6 (SEQ ID

23. A polypeptide according to claim 14, encoded by a nucleotide sequence selected from nucleotides 100 to 3342 of SEQ ID NO:18, or nucleotides 142 to 3468 of SEQ ID A 3 9 5498auspeci 32

24. An isolated and purified plant polypeptide functionally involved in the DNA mismatch repair system of a plant, substantially as hereinbefore described with reference to any one of the examples. An isolated and purified DNA mismatch repair protein active in plants, or a fragment of said protein having the biological activity thereof, wherein said protein or fragment thereof shares at least 50% identity with the amino acid sequence of AtMSH3 (SEQ ID NO: 19) or AtMSH6 (SEQ ID NO: 31).

26. An isolated and purified DNA mismatch repair protein active in plants, or a fragment of said protein having the biological activity thereof, wherein said protein, or fragment thereof is encoded by a nucleotide sequence which shares at least 50% identity with the nucleotide sequence of AtMSH3 (SEQ ID NO: 18) or AtMSH6 (SEQ ID NO:

27. An isolated and purified polypeptide encoded by a DNA polynucleotide according to any one of claims 1 to

28. An isolated and purified polypeptide encoded by a DNA polynucleotide 15 according to any one of claims 11 to 13.

29. An isolated and purified DNA mismatch repair protein active in plants, or a fragment of said protein having the biological activity thereof, substantially as hereinbefore described with reference to any one of the examples, but excluding reference to any non- plant proteins.

30. An isolated and purified DNA polynucleotide comprising a nucleotide sequence selected from the group consisting of: a sequence encoding a polynucleotide which is capable of interfering with the expression of a plant polynucleotide sequence encoding a polypeptide which is homologous to a mismatch repair polypeptide of a yeast or of a human and thereby disabling said plant polynucleotide sequence; and (ii) a sequence 25 encoding a plant polypeptide capable of disrupting the DNA mismatch repair system of a plant; wherein said polypeptide is homologous to AtMSH3 (SEQ ID NO: 19) or to AtMSH6 (SEQ ID NO: 31).

31. A DNA polynucleotide according to claim 30, wherein said plant is a higher plant.

32. A DNA polynucleotide according to claim 31, wherein said plant is selected from members of the Brassicaceae, the Poaceae, the Solanaceae, the Asteraceae, the Malvaceae, the Fabaceae, the Linaceae, the Canabinaceae, the Dauaceae, and the Cucurbitaceae.

33. A DNA polynucleotide according to claim 32, wherein said plant is selected from maize, wheat, oats, barley, rice, tomato, potato, tobacco, capsicum, sunflower, lettuce, artichoke, safflower, cotton, okra, beans, peas, melon, squash, cucumber, oilseed rape, broccoli, cauliflower, cabbage, flax, hemp, hops and carrot. A395498auspcci 33

34. A DNA polynucleotide according to claim 33, wherein said plant is Arabidopsis thaliana. A DNA polynucleotide according to any one of claims 30 to 34 comprising a nucleotide sequence encoding a polynucleotide capable of interfering with the expression of a plant polynucleotide sequence encoding a polypeptide which is homologous to a mismatch repair polypeptide of a yeast or of a human and thereby disabling said plant polynucleotide sequence.

36. A DNA polynucleotide according to claim 35, wherein the encoded polynucleotide is capable of interfering with the expression of a plant polynucleotide sequence is a sense polynucleotide, an antisense polynucleotide or a ribozyme.

37. A DNA polynucleotide according to claim 35 or claim 36, wherein said polypeptide is AtMSH3 (SEQ ID NO: 19) or AtMSH6 (SEQ ID NO: 31).

38. A DNA polynucleotide according to claim 35 or claim 36, wherein said polypeptide shares at least 50% identity with the amino acid sequence of AtMSH3 (SEQ 15 ID NO: 19) or AtMSH6 (SEQ ID NO: 31).

39. A DNA polynucleotide according to claim 35 or claim 36, wherein said plant polynucleotide sequence encoding said polypeptide shares at least 50% identity with the nucleotide sequence ofAtMSH3 (SEQ ID NO: 18) or AtMSH6 (SEQ ID NO: A DNA polynucleotide according to any one of claims 30 to 34 comprising a nucleotide sequence encoding a polypeptide capable of disrupting the DNA mismatch repair system of a plant.

41. A DNA polynucleotide according to claim 40, wherein said polypeptide is homologous to a mismatch repair polypeptide of a yeast or of a human.

42. A DNA polynucleotide according to claim 40, wherein said nucleotide 25 sequence is selected from the polypeptide-encoding portions of gene AtMSH3 (SEQ ID NO: 18) and gene AtMSH6 (SEQ ID

43. A DNA polynucleotide according to claim 40, wherein said nucleotide sequence has a sequence selected from nucleotides 100 to 3342 of SEQ ID NO:18, or nucleotides 142 to 3468 of SEQ ID

44. A DNA polynucleotide according to claim 40, wherein said polypeptide is AtMSH3 (SEQ ID NO: 19) or AtMSH6 (SEQ ID NO: 31). A DNA polynucleotide according to claim 40, wherein said polypeptide shares at least 50% identity with the amino acid sequence of AtMSH3 (SEQ ID NO: 19) or AtMSH6 (SEQ ID NO: 31).

46. A DNA polynucleotide according to claim 40, which shares at least identity with the nucleotide sequence of AtMSH3 (SEQ ID NO: 18) or AtMSH6 (SEQ ID NO: *t. ft. f ft t f ft ft A395498auspecc 34

47. An isolated and purified DNA polynucleotide comprising a nucleotide sequence selected from the group consisting of: a sequence encoding a polynucleotide which is capable of interfering with the expression of a plant polynucleotide sequence encoding a polypeptide which is homologous to a mismatch repair polypeptide of a yeast or of a human and thereby disabling said plant polynucleotide sequence; and (ii) a sequence encoding a polypeptide capable of disrupting the DNA mismatch repair system of a plant; substantially as hereinbefore described with reference to any one of the examples.

48. A recombinant DNA construct comprising a DNA polynucleotide according to any one of claims 40 to 44 or 47 and a regulation element capable of causing overexpression of said polypeptide in a cell of said plant.

49. A recombinant DNA construct comprising a DNA polynucleotide according to claim 45 or claim 46 and a regulation element capable of causing overexpression of said polypeptide in a cell of said plant.

50. A chimeric gene comprising: 15 a DNA polynucleotide according to any one of claims 30 to 47; and at least one regulation element capable of functioning in a plant cell.

51. A chimeric gene according to claim 50 wherein said regulation element is selected from constitutive, inducible, tissue type specific and cell type specific promoters.

52. A chimeric gene according to claim 50 comprising a DNA sequence encoding a polypeptide capable of disrupting the DNA mismatch repair system of a plant, wherein said regulation element is capable of causing overexpression of said polypeptide in a cell of said plant.

53. A chimeric gene according to claim 50 wherein said regulation element is selected from the group consisting of 35S, NOS, PRla, AoPR1 and DMC1.

54. A chimeric gene according to any one of claims 50 to 53, wherein said DNA polynucleotide is a DNA polynucleotide according to any one of claims 35 to 37. A chimeric gene according to any one of claims 50 to 53, wherein said DNA polynucleotide is a DNA polynucleotide according claim 38 or claim 39.

56. A chimeric gene according to any one of claims 50 to 53, wherein said DNA polynucleotide is a DNA polynucleotide according to any one of claims 40 to 44.

57. A chimeric gene according to any one of claims 50 to 53, wherein said DNA polynucleotide is a DNA polynucleotide according claim 45 or claim 46.

58. A chimeric gene comprising: a DNA sequence selected from the group consisting of a sequence encoding a polynucleotide capable of interfering with the expression of a plant polynucleotide sequence encoding a polypeptide which is homologous to a mismatch repair polypeptide of yeast or of a human and thereby disabling said plant polynucleotide sequence, and (ii) a A395498auspeci sequence encoding a polypeptide capable of disrupting the DNA mismatch repair system of a plant; and at least one regulation element capable of functioning in a plant cell; substantially as hereinbefore described with reference to any one of the examples.

59. A plasmid or vector comprising a recombinant DNA construct according to claim 48 or claim 49. A plasmid or vector comprising a chimeric gene according to any one of claims to 58.

61. A plasmid or vector according to claim 59 or claim 60, wherein said DNA polynucleotide is a DNA polynucleotide according to any one of claims 35 to 37.

62. A plasmid or vector according to claim 59 or claim 60, wherein said DNA polynucleotide is a DNA polynucleotide according claim 38 or claim 39.

63. A plasmid or vector according to claim 59 or claim 60, wherein said DNA polynucleotide is a DNA polynucleotide according to any one of claims 40 to 44. 0 15 64. A plasmid or vector according to claim 59 or claim 60, wherein said DNA polynucleotide is a DNA polynucleotide according claim 45 or claim 46.

65. A plasmid or vector comprising: a DNA polynucleotide selected from the group consisting of a sequence encoding a polynucleotide capable of interfering with the expression of a plant polynucleotide sequence encoding a polypeptide which is homologous to a mismatch repair polypeptide of a yeast or of a human and thereby disabling said plant polynucleotide sequence, and (ii) a sequence encoding a polypeptide capable of disrupting the DNA mismatch repair system of a plant; and at least one regulation element capable of functioning in a plant cell; substantially as hereinbefore described with reference to any one of the examples.

66. A plant cell stably transformed, transfected or electroporated with a recombinant DNA construct according to claim 48 or claim 49.

67. A plant cell stably transformed, transfected or electroporated with a DNA polynucleotide according to any one of claims 30 to 47.

68. A plant cell stably transformed, transfected or electroporated with a plasmid or vector according to any one of claims 59 to

69. A plant cell stably transformed, transfected or electroporated with a chimeric gene according to any one of claims 50 to 58. A plant cell according to any one of claims 66 to 69, wherein said DNA polynucleotide is a DNA polynucleotide according to any one of claims 35 to 37.

71. A plant cell according to any one of claims 66 to 69, wherein said DNA polynucleotide is a DNA polynucleotide according claim 38 or claim 39. A395498auspcci 36

72. A plant cell according to any one of claims 66 to 69, wherein said DNA polynucleotide is a DNA polynucleotide according to any one of claims 40 to 44.

73. A plant cell according to any one of claims 66 to 69, wherein said DNA polynucleotide is a DNA polynucleotide according claim 45 or claim 46.

74. A plant cell stably transformed, transfected or electroporated with a DNA polynucleotide selected from: the group consisting of a sequence encoding a polynucleotide capable of interfering with the expression of a plant polynucleotide sequence encoding a polypeptide which is homologous to a mismatch repair polypeptide of a yeast or of a human and thereby disabling said plant polynucleotide sequence; and (ii) a sequence encoding a polypeptide capable of disrupting the DNA mismatch repair system of a plant; or a recombinant DNA construct, a chimeric gene, or a plasmid or vector comprising said DNA polynucleotide, said plant cell being substantially as hereinbefore described with reference to any one of the examples.

75. A plant comprising a cell according to any one of claims 66 to 74. 15 76. A plant according to claim 75, wherein said DNA polynucleotide is a DNA polynucleotide according to any one of claims 35 to 37.

77. A plant according to claim 75, wherein said DNA polynucleotide is a DNA polynucleotide according claim 38 or claim 39.

78. A plant according to claim 75, wherein said DNA polynucleotide is a DNA polynucleotide according to any one of claims 40 to 44.

79. A plant according to claim 75, wherein said DNA polynucleotide is a DNA *0'0 polynucleotide according claim 45 or claim 46. 06" 80. A plant according to any one of claims 75 to 79 selected from members of the Brassicaceae, the Poaceae, the Solanaceae, the Asteraceae, the Malvaceae, the Fabaceae, 25 the Linaceae, the Canabinaceae, the Dauaceae and the Cucurbitaceae.

81. A plant according to any one of claims 75 to 79, wherein said plant is selected from maize, wheat, oats, barley, rice, tomato, potato, tobacco, capsicum, sunflower, lettuce, artichoke, safflower, cotton, okra, beans, peas, melon, squash, cucumber, oilseed rape, broccoli, cauliflower, cabbage, flax, hemp, hops and carrot.

82. A plant comprising one or more cells stably transformed, transfected or electroporated with a DNA polynucleotide selected from: the group consisting of a sequence encoding a polynucleotide capable of interfering with the expression of a plant polynucleotide sequence encoding a polypeptide which is homologous to a mismatch repair polypeptide of a yeast or of a human and thereby disabling said plant polynucleotide sequence; and (ii) a sequence encoding a polypeptide capable of disrupting the DNA mismatch repair system of a plant; or a recombinant DNA construct, a chimeric gene, or a A395498auspeci 37 plasmid or vector comprising said DNA polynucleotide, said plant cell being substantially as hereinbefore described with reference to any one of the examples.

83. A process for at least partially inactivating a DNA mismatch repair system of a plant cell, comprising transforming or transfecting said plant cell with a DNA polynucleotide according to any one of claims 1 to 13 or 30 to 47 and causing said nucleotide sequence to express said polynucleotide or said polypeptide.

84. A process for at least partially inactivating a DNA mismatch repair system of a plant cell, comprising transforming or transfecting said plant cell with a recombinant DNA construct according to claim 48 or claim 49 and causing said nucleotide sequence to express said polynucleotide or said polypeptide. A process for at least partially inactivating a DNA mismatch repair system of a plant cell, comprising transforming or transfecting said plant cell with a chimeric gene according to any one of claims 50 to 58 and causing said nucleotide sequence to express said polynucleotide or said polypeptide. 15 86. A process for at least partially inactivating a DNA mismatch repair system of a plant cell, comprising transforming or transfecting said plant cell with a plasmid or vector according to any one of claims 59 to 65 and causing said nucleotide sequence to express -said polynucleotide or said polypeptide.

87. A process according to any one of claims 83 to 86, wherein said DNA polynucleotide is a DNA polynucleotide according to any one of claims 35 to 37.

88. A process according to any one of claims 83 to 86, wherein said DNA polynucleotide is a DNA polynucleotide according claim 38 or claim 39.

89. A process according to any one of claims 83 to 86, wherein said DNA polynucleotide is a DNA polynucleotide according to any one of claims 40 to 44. 25 90. A process according to any one of claims 83 to 86, wherein said DNA polynucleotide is a DNA polynucleotide according claim 45 or claim 46.

91. A process for at least partially inactivating a DNA mismatch repair system of a plant cell, substantially as hereinbefore described with reference to any one of the examples.

92. A plant cell in which the DNA mismatch repair system has been at least partially inactivated by a process according to any one of claims 83 to 91.

93. A plant comprising one or more cells in which the DNA mismatch repair system has been at least partially inactivated by a process according to any one of claims 83 to 91.

94. A process for increasing genetic variation in a plant comprising obtaining a hybrid plant from a first plant and a second plant, or cells thereof, said first and second plants being genetically different; altering the mismatch repair system in said hybrid plant A395498auspcci 38 by altering the expression of an MSH3 gene and/or an MSH6 gene of said plant by introducing into said plant, or one or more cells thereof, a DNA polynucleotide according to any one of claims 1 to 13 or 30 to 47, or a recombinant DNA construct, chimeric gene or plasmid or vector comprising said DNA polynucleotide; permitting said hybrid plant to self-fertilise and produce offspring plants; and screening said offspring plants for plants in which homeologous recombination has occurred. A process for increasing genetic variation in a plant comprising obtaining a hybrid plant from a first plant and a second plant, or cells thereof, said first and second plants being genetically different; wherein the expression of an MSH3 gene is altered in io said first plant and the expression of an MSH6 gene is altered in said second plant, said gene expression alterations being introduced into said plants, or one or more cells thereof, by inserting into said plants a DNA polynucleotide according to any one of claims 1 to 13 or 30 to 47, or a recombinant DNA construct, chimeric gene or plasmid or vector comprising said DNA polynucleotide; permitting said hybrid plant to self-fertilise and 15 produce offspring plants; and screening said offspring plants for plants in which homeologous recombination has occurred. S-96. A process according to claim 95, wherein an MSH3 gene is incapacitated in said first plant, and a MSH6 gene is incapacitated in said second plant, and said MSH3 and MSH6 genes are incapacitated in said hybrid plant thereby altering the mismatch repair system of said hybrid plant. 0.

97. A process according to claim 96, wherein said incapacitation of the mismatch repair system of said offspring plants is reversible.

98. A process according to claim 97, wherein the mismatch repair system in resulting offspring plants is restored.

99. A process according to claim 94 or 95, wherein said incapacitation of the S-mismatch repair system of said offspring plants is reversible.

100. A process according to claim 99, wherein the mismatch repair system in resulting offspring plants is restored.

101. A process according to any one of claims 94 to 100, wherein a new genetic linkage of a desired characteristic trait or of a gene which contributes to a desired characteristic trait is observable in at least one of said offspring plants.

102. A process for increasing genetic variation in a plant, substantially as hereinbefore described with reference to any one of the examples.

103. A plant comprising one or more cells in which the DNA mismatch repair system has been altered, and optionally restored, obtained by a process according to any one of claims 94 to 102.

104. A process for obtaining a plant having a desired characteristic, comprising altering the mismatch repair system in a plant, cell or plurality of cells of a plant which A395498auspcci 39 does not have said desired characteristic, permitting mutations to persist in said cells to produce mutated plant cells, deriving plants from said mutated plant cells, and screening said plants for a plant having said desired characteristic, wherein said mismatch repair system is altered by altering the expression of an MSH3 gene and/or an MSH6 gene of said plant by introducing into said plant, cell or plurality of cells, a DNA polynucleotide according to any one of claims 1 to 13 or 30 to 47, or a recombinant DNA construct, chimeric gene or plasmid or vector comprising said DNA polynucleotide.

105. A process according to claim 104 wherein said step of altering the mismatch repair system comprises introducing into said hybrid plant, plant, cell or cells a chimeric gene according to any one of claims 50 to 58 and permitting the chimeric gene to express a polynucleotide which is capable of interfering with the expression of a plant polynucleotide sequence in a mismatch repair gene of the hybrid plant, cell or cells, or a polypeptide capable of disrupting the DNA mismatch repair system of the hybrid plant, cell or cells.

106. A process according to claim 104 or claim 105 comprising inactivating an 15 MSH3 gene and/or an MSH6 gene of said plant.

107. A process according to claim 104 or claim 105 comprising inactivating an MSH3 gene and an MSH6 gene of said plant.

108. A process according to claim any one of claims 104 to 107 comprising at least partially inactivating the mismatch repair system of said plant in a predetermined cell type or in a predetermined tissue of said plant.

109. A process according to any one of claims 104 to 108 further comprising :restoring mismatch repair in said plant, cell or cells. o,

110. A process for obtaining a plant having a desired characteristic, comprising altering the mismatch repair system in a plant, cell or plurality of cells of a plant, said method being substantially as hereinbefore described with reference to any one of the examples. .o0 111. A plant having a desired characteristic and comprising one or more cells in which the DNA mismatch repair system has been altered, and optionally restored, obtained by a method according to any one of claims 104 to 110.

112. A DNA polynucleotide according to any one of claims 1 to 10, when used for at least partially inactivating a DNA mismatch repair system of a plant cell.

113. A DNA polynucleotide according to any one of claims 11 to 13, when used for at least partially inactivating a DNA mismatch repair system of a plant cell.

114. A DNA polynucleotide according to any one of claims 30 to 47, when used for at least partially inactivating a DNA mismatch repair system of a plant cell.

115. A DNA polynucleotide when used according to claim 114, wherein said DNA polynucleotide is a DNA polynucleotide according to any one of claims 35 to 37.

116. A DNA polynucleotide when used according to claim 114, wherein said DNA polynucleotide is a DNA polynucleotide according claim 38 or claim 39. A395498auspci

117. A DNA polynucleotide when used according to claim 114, wherein said DNA polynucleotide is a DNA polynucleotide according to any one of claims 40 to 44.

118. A DNA polynucleotide when used according to claim 114, wherein said DNA polynucleotide is a DNA polynucleotide according claim 45 or claim 46.

119. A DNA polynucleotide when used for at least partially inactivating a DNA mismatch repair system of a plant cell, substantially as hereinbefore described with reference to any one of the examples.

120. A DNA polynucleotide according to any one of claims 1 to 10, when used for increasing genetic variation in a plant.

121. A DNA polynucleotide according to any one of claims 11 to 13, when used for increasing genetic variation in a plant.

122. A DNA polynucleotide according to any one of claims 30 to 47, when used for increasing genetic variation in a plant.

123. A DNA polynucleotide when used according to claim 122, wherein said DNA 15 polynucleotide is a DNA polynucleotide according to any one of claims 35 to 37.

124. A DNA polynucleotide when used according to claim 122, wherein said DNA polynucleotide is a DNA polynucleotide according claim 38 or claim 39.

125. A DNA polynucleotide when used according to claim 122, wherein said DNA polynucleotide is a DNA polynucleotide according to any one of claims 40 to 44.

126. A DNA polynucleotide when used according to claim 122, wherein said DNA polynucleotide is a DNA polynucleotide according claim 45 or claim 46. *:127. A DNA polynucleotide when used for increasing genetic variation in a plant, substantially as hereinbefore described with reference to any one of the examples.

128. A DNA polynucleotide according to any one of claims 1 to 10, when used for 25 obtaining a plant having a desired characteristic.

129. A DNA polynucleotide according to any one of claims 11 to 13, when used for obtaining a plant having a desired characteristic.

130. A DNA polynucleotide according to any one of claims 30 to 47, when used for obtaining a plant having a desired characteristic.

131. A DNA polynucleotide when used according to claim 130, wherein said DNA polynucleotide is a DNA polynucleotide according to any one of claims 35 to 37.

132. A DNA polynucleotide when used according to claim 130, wherein said DNA polynucleotide is a DNA polynucleotide according claim 38 or claim 39.

133. A DNA polynucleotide when used according to claim 130, wherein said DNA polynucleotide is a DNA polynucleotide according to any one of claims 40 to 44.

134. A DNA polynucleotide when used according to claim 130, wherein said DNA polynucleotide is a DNA polynucleotide according claim 45 or claim 46.

135. A DNA polynucleotide when used for obtaining a plant having a desired characteristic, substantially as hereinbefore described with reference to any one of the examples. A395498auspeci

136. An oligonucleotide capable of hybridising at 60 0 C under standard PCR conditions to the DNA of SEQ ID NO: 18 with the proviso that said oligonucleotide is other than SEQ ID NO: 1 or SEQ ID NO:2.

137. The oligonucleotide of claim 136, wherein said oligonucleotide is selected from the group consisting of SEQ ID NOs: 3, 6 to 11, 13, 14, 16 and 17.

138. The oligonucleotide of claim 136 or claim 137, which is capable of hybridising at 65 0 C under standard PCR conditions to the DNA of SEQ ID NO: 18.

139. An oligonucleotide capable of hybridising at 45C under standard PCR conditions to the DNA of SEQ ID NO:30 with the proviso that said oligonucleotide is other than SEQ ID NO:1 or SEQ ID NO:2.

140. The oligonucleotide of claim 139, wherein said oligonucleotide is selected from the group consisting of SEQ ID NOs: 19 to 25, 28 or 29.

141. The oligonucleotide of claim 139 or claim 140, which is capable of hybridising at 50 0 C under standard PCR conditions to the DNA of SEQ ID NO: 15 142. The oligonucleotide of claim 139 or claim 140, which is capable of hybridising at 55°C under standard PCR conditions to the DNA of SEQ ID NO:

143. The oligonucleotide of claim 139 or claim 140, which is capable of hybridising at 60 0 C under standard PCR conditions to the DNA of SEQ ID NO:

144. The oligonucleotide of claim 139 or claim 140, which is capable of hybridising at 65 0 C under standard PCR conditions to the DNA of SEQ ID NO:

145. The oligonucleotide according to any one of claims 136 to 138, when used for detecting or isolating a plant MSH3 homolog.

146. The oligonucleotide according to any one of claims 139 to 144, when used for detecting or isolating a plant MSH6 homolog. 25 Dated 23 April, 2002 Aventis CropScience S.A. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON A395498auspcci