US20040205839A1

US20040205839A1 - Methods for obtaining plant varieties

Info

Publication number: US20040205839A1
Application number: US10/741,789
Authority: US
Inventors: Marie-Pascale Doutriaux; Andreas Betzner; Georges Freyssinet; Pascal Perez
Original assignee: Individual
Current assignee: Individual
Priority date: 1997-10-10
Filing date: 2003-12-19
Publication date: 2004-10-14
Also published as: ZA989242B; WO1999019492A2; AUPO974597A0; EP1027447A2; CA2305817A1; WO1999019492A3; US6734019B1; AR017320A1

Abstract

The present invention relates to an isolated and purified DNA comprising a nucleotide sequence that encodes a polypeptide functionally involved in the DNA mismatch repair system of a plant.

Description

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 (e.g. growth performance, yield. etc.) or qualitative (e.g. 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: 30), 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% 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 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×E. coli occurs five orders of magnitude more frequently than homeologous recombination in conjugational crosses E. coli×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 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 25, 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 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), mouse (mPMS2) and human (hMLH1 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

According to a first embodiment of the invention, there is provided an isolated and purified DNA molecule comprising a polynucleotide sequence encoding a polypeptide functionally involved in the DNA mismatch repair system of a plant. In one form of this embodiment, the invention provides 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 invention provides polynucleotide sequences encoding polypeptides which are homologous to the mismatch repair polypeptides MSH3 and MSH6 of Saccharomyces cerevisiae. Still more particularly, the invention provides the 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.

According to a second embodiment of the invention, there is provided 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 of Arabidopsis thaliana, as defined hereinbelow.

According to a third embodiment of the invention, there is provided an isolated and purified DNA molecule comprising a polynucleotide sequence selected from the group consisting of (i) 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.

According to a fourth embodiment of the invention there is provided a chimeric gene comprising a DNA sequence selected from the group consisting of (i) 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, PR1a, 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.

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 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 sequence of the third embodiment or a chimeric gene of the fourth embodiment or a plasmid or vector of the fifth embodiment, and causing said DNA sequence to express said polynucleotide or said polypeptide.

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; 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.

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 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.

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 a polypeptide capable of disrupting the DNA mismatch repair system of the hybrid plant or cells.

In other embodiments, the invention provides (a) an oligonucleotide capable of hybridising at 45° C. under standard PCR conditions to a DNA molecule of the first embodiment; (b) an oligonucleotide capable of hybridising at 45° C. under standard PCR conditions to the DNA of SEQ ID NO: 18 and (c) an oligonucleotide capable of hybridising at 45° C. under standard PCR conditions to the DNA of SEQ ID NO:30; with the proviso that the oligonucleotide of (a), (b) and (c) is other than SEQ ID NO:1 or SEQ ID NO:2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a diagrammatic representation of the primer sequences used to isolate AtMSH3. [0031]
FIG. 2 is a plasmid map of [0032] clone 52, showing restriction enzyme cleavage sites in the 5′ half of the full-length cDNA for AtMSH3.
FIG. 3 is a plasmid map of [0033] clone 13, showing restriction enzyme cleavage sites in the 3′ half of the full-length cDNA for AtMSH3.
FIG. 4 is a sequence listing of the coding sequence of AtMSH3, together with a deduced sequence of the encoded polypeptide. [0034]
FIG. 5 is a protein alignment of yeast ([0035] Saccharomyces cerevisiae) and Arabidopsis thaliana MSH3 protein.
FIG. 6 provides a diagrammatic representation of the primer sequences used to isolate AtMSH6. [0036]
FIG. 7 is a plasmid map of [0037] clone 43, showing restriction enzyme cleavage sites in the 5′ half of the full-length cDNA for AtMSH6.
FIG. 8 is a plasmid map of [0038] clone 62, showing restriction enzyme cleavage sites in the 3′ half of the full-length cDNA for AtMSH6.
FIG. 9 is a sequence listing of the coding sequence of AtMSH6, together with a deduced sequence of the encoded polypeptide. [0039]
FIG. 10 is a protein alignment of yeast ([0040] Saccharomyces cerevisiae) and Arabidopsis thaliana MSH6 protein.
FIG. 11 is a genomic sequence listing of AtMSH6. [0041]
FIG. 12 is a plasmid map of plasmid pPF13. [0042]
FIG. 13 is a plasmid map of plasmid pPF14. [0043]
FIG. 14 is a plasmid map of plasmid pCW 186. [0044]
FIG. 15 is a plasmid map of plasmid pCW187. [0045]
FIG. 16 is a plasmid map of plasmid pPF66. [0046]
FIG. 17 is a plasmid map of plasmid pPF57. [0047]
FIG. 18 is a diagrammatic representation of an antisense gene construction for use in homeologous meiotic recombination. [0048]
FIG. 19 is a plasmid map of plasmid p3243.[0049]

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 [0050] E. coli, and to MMR genes in yeasts and humans.
Thus, the inventors have identified genes, herein designated AtMSH3 and AtMSH6, of the plant [0051] 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 polypeptides homologous to yeast MSH2. MLH1 or PMS2, or to human MLH1. PMS1 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, artichoke, safflower, cotton, okra, beans of many kinds including soybean, peas, melon, squash, cucumber, oilseed rape, broccoli, cauliflower, cabbage, flax, hemp, hops and carrot. [0052]
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. [0053]
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 [0054] 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° 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° 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° 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° C., for example at least 50° C., or at least 55° C. or at least 60° C. or as high as 65° C.
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, MSH5, PMS1. PMS2 and MLH1 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. [0055]
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 [0056] 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 [0057] 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 (e.g. callus; embryonic tissue; etc.), cell type specific (e.g. protoplasts; meiocytes; etc.) or developmental (e.g. embryo) expression of the altering gene or genes, in order for the 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 PR1a (reviewed by Gatz, 1997, Annual Rev. Plant Phys. Plant Mol. Biol. 48: 89), tissue specific promoters such as AoPR1 (Sabahattin et al., 1993, Biotechnology 11: 218), and cell-type specific promoters such as DMC1. [0058]
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 rate, flowering time, ripening time, etc. [0059]
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 site-specific mutagenesis. [0060]
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 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 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. [0061]
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 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. [0062]
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 (e.g. oil, starch, protein), altered composition or quality of cell walls (e.g. decrease in lignin content), altered growth rate, prolonged flowering, increased plant yield or grain yield, altered plant morphology, resistance to pathogens, tolerance to or improved performance under environmental stresses of various kinds, etc. [0063]
In a preferred form of the tenth embodiment, an MMR altering gene is co-introduced 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 co-introduced 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 include both the MSH3 and MSH6 altering genes and its MMR system will therefore be inactivated. [0064]
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 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 specific or cell type specific promoter which only renders the MMR altering gene active under limited conditions. [0065]
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 [0066] 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 [0067] 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. [0068]

EXAMPLES

Example 1

Cloning of the AtMSH3 and AtMSH6 Coding Sequences

Isolation of Partial AtMSH3 and AtMSH6 Consensus Sequences [0069]
Degenerate oligonucleotides UPMU (SEQ ID NO:1) and DOMU (SEQ ID NO.2) [0070]

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. [0071]
Primers UPMU and DOMU correspond to conserved amino acid sequences of the proteins MutS ([0072] E. coli and S. typhimurium), HexA (S. pneumoniae). Rep1 (mouse) and Duc1 (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 for DOMU (amino acid positions 964-968 for AtMSH6 and 928-932 for AtMSH3, respectively.) These primers have been used to isolate MSH2 and MSH1 from yeast (Reenan and Kolodner. Genetics 132: 963-97 (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 μg total RNA from a cell suspension culture of [0073] Arabidopsis thaliana ecotype Columbia (Axelos et at. 1989, Mol. Gen. Genetics 219: 106-112). The PCR reaction was performed under the following conditions in a final volume of 100 μl: 0.2 mM dNTP, 1 μM each primer, 1×PCR buffer, 1 u 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° C., 90 seconds at 45° C., 1 minute at 72° C. The amplification products were cloned into pGEM-T vector (Promega) and sequenced. Two different clones were isolated, S5 (350 bp) was homologous to MSH3, S8 (327 bp) 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 2 μg 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 5′ and 3′ RACE PCR reactions in the presence of primers that are specific for the adaptor on one side (AP1 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 [0074]
From the sequence of clone S5, primer 636 (SEQ ID NO:3) was designed: [0075]

636 TGCTAGTGCCTCTTGCAAGCTCAT.
Primer AP1 (SEQ ID NO:4) is complementary to a portion of an adaptor sequence which had been ligated to the 5′ and 3′ ends of [0076] Arabidopsis cDNA:

AP1 CCATCCTAATACGACTCACTATAGGGC.
PCR performed on the ligated cDNA with [0077] primers 636 and AP1 for the 5′ RACE PCR was followed by a second round of amplification with the nested primers AP2 (SEQ ID NO:5) 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 2720 bp DNA fragment. FIG. 1 provides a diagrammatic representation of the primer sequences used to isolate AtMSH3. Another primer (S51, SEQ ID NO:7) [0078]

S51 GGATCGGGTACTGGGTTTTGAGTGTGAGG
was designed closer to the 5′ border and permitted the determination of 99 bp upstream to the ATG initiation codon. For the 3′ RACE PCR, a first PCR reaction was performed with primers API and 635 (SEQ ID NO:8). [0079]

635 GCACGTGCTTGATGGTGTTTTCAC
followed by a second round of amplification, using the nested primers AP2 and S523 (SEQ ID NO:9) [0080]

S523 TCAGACAGTATCCAGCATGGCAGAAGTA
which produced a DNA fragment of 890 bp. Both DNA fragments were subcloned into pGEM-T and sequenced. Since PCR amplification using the Expand Long Template PCR 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) [0081]

1S5 ATCCCGGGATGGGCAAGCAAAAGCAGCAGACGA

S53 GACAAAGAGCGAAATGAGGCCCCTTGG
amplified the 1244 bp fragment clone 52 (SEQ ID NO:12′, cloned into pUC18SmaI). PCR with primers S52 (SEQ ID NO:13) and 2S5 (SEQ ID NO:14) [0082]

2S5 ATCCCGGGTCAAAATGAACAAGTTGGTTTTAGTC

S52 GCCACATCTGACTGTTCAAGCCCTCGC
amplified the 2104 bp clone 13 (SEQ ID NO:15, cloned into pUC18/SmaI). 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 SmaI fragment from pPF26 contains the complete AtMSH3 coding sequence. The remaining primers referred to in FIG. 1 are as follows: [0083]

S51 GGATCGGGTACTGGGTTTTGAGTGTGAGG (SEQ ID NO:16)

S525 AGGTTCTGATTATGTGTGACGCTTTACTTA (SEQ ID NO:17)
FIGS. 2 and 3 provide plasmid maps of [0084] clones 52 and 13 respectively, showing restriction enzyme cleavage sites. The complete AtMSH3 coding sequence (SEQ ID NO:18) is 3246 bp long and is shown in FIG. 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 FIG. 5.
Isolation of the AtMSH6 Complete Coding Sequence and Genomic Sequences [0085]
The same procedure allowed isolation of the AtMSH6 cDNA. FIG. 6 provides a diagrammatic representation of the primer sequences used to isolate AtMSH6. For the 5′ RACE PCR, primers 638 (SEQ ID NO:20) and API (SEQ ID NO:4) [0086]

638 TCTCTACCAGGTGACGAAAAACCG
allowed the amplification of a 2889 DNA fragment. Primer S81 (SEQ ID NO:21) [0087]

S81 CGTCGCCTTTAGCATCCCCTTCCTTCAC
helped define the 142 bp upstream to the ATG initiation codon. On the 3′ side. RACE PCR was initially performed with primers S82′3 (SEQ ID NO:22) and AP1 (SEQ ID NO:4), [0088]

S823 GCTTGGCGCATCTAATAGAATCATGACAGG
and then with the nested primers 637 (SEQ ID NO:23) and AP2 (SEQ ID NO:5). [0089]

637 GACAGCGTCAGTTCTTCAGAATGC

to produce a 774 bp 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/SmaI), and a 1379 bp clone identified as clone 62 (SEQ ID NO:27, also cloned in pUC18/SmaI).


1S8	ATCCCGGGATGCAGCGCCAGAGATCGATTTTGT

2S8	ATCCCGGGTTATTTGGGAACACAGTAAGAGGATT	(SEQ ID

		NO: 28)

S82	GCGTTCGATCATCAGCCTCTGTGTTGC	(SEQ ID

		NO: 29)

S83	CGCTATCTATGGCTGCTTCGAATTGAG

FIGS. 7 and 8 provide plasmid maps of [0091] clones 43 and 62 respectively, showing restriction enzyme cleavage sites. Clones 43 and 62 were digested by the Xmn1 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 3330 bp long and is shown in FIG. 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 FIG. 10.
An AtMSH6 genomic sequence was also isolated from a genomic DNA library constituted after partial Sau3AI digestion of DNA from the [0092] Arabidopsis cell suspension. 8062 bp 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 FIG. 11.

Example 2

A Measure of Somatic Variation in MMR Deficient Plants

Constructs [0093]
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 [0094] Escherichia coli or Agrobacterium tumefaciens bacteria. The aacC1 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 35S promoter/terminator cassette with a polylinker was introduced into pPZP121. 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 (2104 bp), for AtMSH6 this corresponds to clone 62 (1379 bp). Clone 13 comprises 2104 bp of the 3′ region that were cut off the pUC18 vector by SalI/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 (1379 bp) 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 reciprocally, the 3′ region of AtMSH3 (from clone 13) was introduced ahead of AtMSH6 into pPF14, creating pCW187.
These constructs were introduced into the Arabidopsis cells (as described below) of wildtype Columbia and of the Columbia tester line. [0095]
An alternative strategy to antisense inhibition of AtMSH6 comes from experiments of Marra et al. (1998. Proc. Natl. [0096] 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 SmaI, 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. FIGS. 12-16 provide plasmid maps of plasmids pPF13, pPF14, pCW186, pCW187 and pPF66, respectively. [0097]
Construction of Tester Construct [0098]
For the purpose of Forward Mutagenesis Assays, a tester construct was built containing the coding regions for nptII, codA, uidA. All three genes are driven by the 35S promoter and are terminated by the 35S terminator. This construct was obtained by introducing an EcoRI fragment encoding the codA cassette (2.5 kb) and a HindIII fragment encoding the uidA (GUS) cassette (2.4 kb) into the pPZP111 vector (Hajdukiewicz et al., 1994, Plant Mol Biol 23: 793-799) which already contained the nptII 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 (i.e. to identify cell lines in which the transgenes are expressed), and codA is used as a marker for forward mutagenesis (described below). [0099]
The plasmid map of pPF57 is provided in FIG. 17. [0100]
Plant Cell Transformation [0101]
The constructs are introduced into [0102] Agrobacterium 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 B5 medium. 30 g/l sucrose, 200 μg/l NAA). 10 μl of saturated Agrobacterium containing the transforming T-DNA constructs are added to 10 ml diluted cells in a 100 ml erlenmeyer. The co-cultivation is agitated slowly (80 rpm) for 2 days. The cells are then washed 3 times into AT medium and finally resuspended in the same initial volume (10 ml). The culture is agitated for 3 days to allow expression before plating on selection plates (AT/BactoAgar 8 g/l+gentamycin 50 μg/ml). Transformed individual calli are isolated 3 weeks later.
Tester Strain [0103]
The tester construct on plasmid pPF57 was introduced into [0104] Arabidopsis cells of 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 5-fluoro-cytosine (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. [0105]
Microsatellite Analysis [0106]
Microsatellites have been described in [0107] 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 P³²labelled 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[0108] ₁₈), ngaI72 (GA₂₉), and ATHGENEA (A₃₉), 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 rapidly extracted from the calli and is analysed for microsatellite instability as described in detail by Bell and Ecker 1994, [0109] 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 ngaI72 are included in Table 1. PCR amplification of microsatellite ATHGENEA made use of a forward primer containing the sequence [0110]

ACCATGCATAGCTTAAACTTCTTG (SEQ ID NO: 32)
and of a reverse primer containing the sequence [0111]

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 length of 124 bp. DNA sequencing verified this fragment as a CA[0112] ₁₈microsatellite.
Forward Mutagenesis Assay [0113]
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 5FC are first analysed for GUS activity. If GUS is inactive, 5FC resistance is assumed to be due to gene silencing (all three genes are under the 35S promoter). If GUS is active, 5FC 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. [0114]
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). [0115]

Example 3

Homeologous Meiotic Recombination in Arabidopsis Thaliana

A. Construction of a Meiocyte Specific Gene Expression Cassette Comprising the DMC1 Promoter and the NOS Terminator [0116]
(i) 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 long subfragment of the DMC1 promoter was obtained by PCR from genomic DNA of [0117] Arabidopsis thaliana (ssp. Landsberg erecta “Ler”).
The PCR was done in three rounds: [0118]
Round One: A 3.7 Kb long product was obtained using the forward primer DMCIN-A comprising the sequence [0119]

GAAGCGATATTGTTCGTG (SEQ ID NO: 34)
and the reverse primer DMCIN-B comprising the sequence [0120]

AGATTGCGAGAACATTCC. (SEQ ID NO: 35)
The weak amplification product was then used as template for round two and three. [0121]
Round Two: A 3.1 Kb long product comprising the promoter and the 5′ untranslated leader was obtained using forward primer DMCIN-1, which contained the sequence [0122]

acgcgtcgacTCAGCTATGAGATTACTCGTG (SEQ ID NO :36)
and introduced a SalI cloning site at the 5′ end of the promoter fragment, and reverse primer DMCIN-2 which contained the sequence [0123]

gctctagaTTTCTCGCTCTAAGACTCTCT (SEQ ID NO:37)
and introduced a XbaI site at the 3′ end of the PCR fragment. [0124]
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 [0125]

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 [0126]

tcccccgggctcgagagatctccatggTTTCTTCAGCTCTATGAATCC (SEQ ID NO:39)
and introduced at the 3′ end of the PCR product restriction sites for NcoI. BglII, XhoI and SmaI. [0127]
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 SalI and SmaI of p3264, which contains the SacI-EcoRI NOS terminator in pBIN19, to yield the entire expression cassette in pBIN19. This cassette contains the following cloning sites: NcoI, BglII, XhoI. SmaI and (already present on p3264) KpnI and SacI. [0128]
(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 [0129] Arabidopsis thaliana (ssp. Landsberg erecta “Ler”). The forward PCR primer (DMC1a) contained the sequence

acgcgtcgacGAATTCGCAAGTGGGG (SEQ ID NO:40)
and introduced a SalI cloning site at the 5′ end of the promoter fragment. The reverse PCR primer (DMC1b) contained the sequence [0130]

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° C., 1 minute at 54° C., 2 minutes at 72° C. [0131]
The PCR reaction yielded a blunt ended DNA fragment which was digested with restriction enzyme SalI and was cloned into the cleavage sites of restriction enzymes SalI and SmaI in plasmid p2030, a pUC118 derivative containing the SacI-EcoRI NOS terminator fragment of pBIN121. The cloning yielded plasmid p2031, containing the DMC1 promoter-polylinker-NOS terminator expression cassette depicted in FIG. 18. [0132]
B. Construction of an MSH3 Antisense Gene Under the Control of the DMC1 Promoter [0133]
A 2.1 kb DNA fragment encoding the carboxyterminal part of AtMSH3 was removed from the polylinker of [0134] clone 13 described in Example 1 after (i) 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 is promoter in plasmid p2031, which had been digested with restriction enzymes SmaI and BglII. This cloning yielded plasmid p2033 (FIG. 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 (FIG. 18). [0135]
C. Construction of a Combined MSH6/MSH3 Antisense Gene Under the Control of a Single DMC1 Promoter [0136]
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 FIG. 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. [0137]
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 [0138] 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 cloning, the recipient plasmid may be cleaved with either AvaI or NcoI 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 SacI. 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 [0139]
Overexpression of MSH3 protein was shown in human cells (Marra et al., 1998, Proc. Natl. [0140] 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 [0141] Arabidopsis as an alternative to antisense inhibition of AtMSH6. For this purpose the full-length 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 [0142] Arabidopsis Thaliana Subspecies Landsbere Erecta (Ler). Columbia (Col) and C24, Respectively
E (i). Visual Recombination Markers in [0143] 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 [0144] 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 [0145] 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 on Chr.4, and ms1-1 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. [0146]
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: [0147]
NW22: relative markers are dis1-(4 cM)-ga4-(11 cM)-th1 on [0148] chromosome 1.
NW10: relevant markers are tz-201-(5 cM)-cer3 on [0149] chromosome 5.
NW134, relevant markers are ttg-(4 cM)-ga3 on [0150] chromosome 5.
NW24 (abi3-1) and NW64 (gl1-1). When present in the same plant on [0151] chromosome 3, abi3-1 and gl1-1 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: [0152]
The genome of [0153] 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 (25 SSLPs), respectively. Polymorphic SSLPs can be used as molecular markers in the analysis of homeologous recombination between genomes from these subspecies. [0154]
The PCR reactions (25 μL) were carried out over 35 cycles (15 seconds at 94° C., 30 seconds at 55° C. and 30 seconds at 72° C.), with 0.25 U Taq DNA polymerase and 0.6 μg genomic DNA in reaction buffer containing 2 mM MgCl[0155] ₂. PCR products were separated by agarose gel electrophoresis (4% ultra high resolution agarose) and visualised by ethidiumbromide staining. The results from the PCR experiments are summarised in Table 1, which also shows the sequence of PCR primers, primer annealing temperature (Tm), PCR product length and chromosome location of SSLP (with respect to the R1 map of May 1997, Weeds World 4i).
F. Production of Hybrid Plants [0156]
C24 plants heterozygous for chlorina3A:AclGUS are crossed as male to emasculated wildtype Ler to produce Ler/C24(chlorina3A, GUS) hybrid seeds. [0157]
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 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. [0158]
Ler plants homozygous for the five chromosome markers are male sterile (ms1-1) and are crossed without emasculation to wildtype C24 to produce Ler (an-1, py-1, gl1-1, cer2-1, ms1-1) C24 hybrid seeds. [0159]
Other Ler plants, which are male fertile, are crossed after emasculation of the female parent to produce Ler/C24 hybrid seeds. [0160]
G. Introduction of MSH3 and MSH6/3 Antisense Genes into Arabidopsis and Analysis of Meiotic Homeologous Recombination [0161]
(i) Transformation of Hybrid Plants and Analysis of Homeologous Meiotic Recombination [0162]
The plant transformation vectors comprising the antisense genes described in (B) and (C) above are introduced into [0163] Agrobacterium tumefaciens 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 [0164] Arabidopsis subspecies described in (F). 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 (B) and (C) above, leading to decreased MSH6 and/or MSH3 gene 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. [0165]
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 (E) above. [0166]
In case of homeologous recombination between chromosomes of Ler and C24(chlorina3A:Ac, GUS), the analysis concentrates on [0167] 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 Wildtype, Hybrid Plant Production and Analysis of Homeologous Meiotic Recombination [0168]
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). [0169]
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. [0170]
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 (F). 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 [0171] DMC 1 promoter is activated and can direct the expression of antisense or sense genes described in (B), (C) and (D) above, leading to decreased MSH6 and/or MSH3 gene 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 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. [0172]
Examples of recombination analysis are the following. [0173]
The Ler line NW22(dis1, ga4, th1) is used for crosses to transformedC24. [0174]
F2 plants are preselected first for thiamine requirement (th1) and then are further analysed for re-appearance of wildtype height (loss of ga4) and/or re-appearance of normal trichomes (loss of dis1) as a result of recombination. [0175]
The Ler line NW10(tz-201, cer3) is used for crosses to transformedC24. [0176]
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. [0177]
The Ler line NW134 (ttg, 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. [0178]
Ler plants homozygous for abi3-1 and gl1-1 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. [0179]

In the case of homeologous recombination between transformedC24 and the Ler line NW1 (an-1, py-1, gl1-1, cer2-1, ms1-1), 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.

TABLE 1


SSLP Markers in Arabidopsis thaliana Subspecies

	RI Map	PCR Primer
Chromosome	Position	Pair	Primer Sequence	Tm [° C.]	length/COL	length/LER	length/C24

I	2.3	ATEAT1 F	GCCACTGCGTGAATGATATG	57.8	172	162	162

		ATEAT1 R	CGAACAGCCAACATTAATTCCC	58.2

I	9.3	NGA63 F	AACCAAGGCACAGAAGCG	57.3	111	89	120

		NGA63 R	ACCCAAGTGATCGCCACC	59.6

I	40.1	NGA248 F	TACCGAACCAAAACACAAAGG	56.1	143	129	no amplific.

		NGA248 R	TCTGTATCTCGGTGAATTCTCC	58.2

I	81.2	NGA128 F	GGTCTGTTGATGTCGTAAGTCG	60.1	180	190	no amplific.

		NGA128 R	ATCTTGAAACCTTTAGGGAGGG	58.2

I	81.2	NGA280 F	CTGATCTCACGGACAATAGTGC	60.1	105	85	85

		NGA280 R	GGCTCCATAAAAAGTGCACC	57.8

I	111.4	NGA111 F	CTCCAGTTGGAAGCTAAAGGG	60	128	162	170

		NGA111 R	TGTTTTTTAGGACAAATGGCG	70

II	ca. 7.5	NGA168 F	CCTTCACATCCAAAACCCAC	57.8	213	217	208

		NGA168 R	GCACATACCCACAACCAGAA	57.8

II	ca. 48	NGA1126L	CGCTACGCTTTTCGGTAAAG	57.8	191	199	196

		NGA1126R	GCACAGTCCAAGTCACAACC	59.9

II	62.2	NGA361L	AAAGAGATGAGAATTTGGAC	51.7	114	120	114

		NGA361R	ACATATCAATATATTAAAGTAGC	49.5

II	73	NGA168 F	TCGTCTACTGCACTGCCG	59.6	151	135	135

		NGA168 R	GAGGACATGTATAGGAGCCTCG	61.9

II	ca. 77	AthBIO2 L	TGACCTCCTCTTCCATGGAG	59.9	141	209	139

		AthBIO2 R	TTAACAGAAACCCAAAGCTTTC	54.5

II	ca. 83	AthUBIQUE L	AGGCAAATGTCCATTTCATTG	54.1	146	148	148

		AthUBIQUE R	ACGACATGGCAGATTTCTCC	57.8

III	3.4	NGA172 F	AGCTGCTTCCTTATAGCGTCC	60	162	136	140

		NGA172 R	CATCCGAATGCCATTGTTC	55.4

III	12.8	NGA126 F	GAAAAAACGCTACTTTCGTGG	56.1	119	147	no amplific.

		NGA126 R	CAAGAGCAATATCAAGAGCAGC	58.2

III	17.5	NGA162 F	CATGCAATTTGCATCTGAGG	55.8	107	89	no amplilic.

		NGA162 R	CTCTGTCACTCTTTTCCTCTGG	60.1

III	81.8	NGA6 F	TGGATTTCTTCCTCTCTTCAC	56.1	143	123	143

		NGA6 R	ATGGAGAAGCTTACACTGATC	56.1

IV	19.8	NGA12 F	AATGTTGTCCTCCCCTCCTC	59.9	247	234	220

		NGA12 R	TGATGCTCTCTGAAACAAGAGC	58.2

IV	24.1	NGA8 F	GAGGGCAAATCTTTATTTCGG	56.1	154	198	190

		NGA8 R	TGGCTTTCGTTTATAAACATCC	54.5

IV	102	NGA1107 L	GCGAAAAAACAAAAAAATCCA	50.2	150	140	140

		NGA1107 R	CGACGAATCGACAGAATTAGG	58

V	11.8	NGA225 F	GAAATCCAAATCCCAGAGAGG	58	119	189	119

		NGA225 R	TCTCCCCACTAGTTTTGTGTCC	60.1

V	16.7	NGA249 F	TACCGTCAATTTCATCGCC	55.4	125	115	115

		NGA249 R	GGATCCCTAACTGTAAAATCCC	58.2

V	19.9	CA72 F	AATCCCAGTAACCAAACACACA	56.3	124	110	110

		CA72 R	CCCAGTCTAACCACGACCAC	61.9

V	20	NGA151 F	GTTTTGGGAAGTTTTGCTGG	55.8	150	120	130

		NGA151 R	CAGTCTAAAAGCGAGAGTATGATG	58.6

V	24	NGA106 F	GTTATGGAGTTTCTAGGGCACG	60.1	157	123	130

		NGA106 R	TGCCCCATTTTGTTCTTCTC	55.8

V	37.8	NGA139 F	AGAGCTACCAGATCCGATGG	59.9	174	132	132

		NGA139 R	GGTTTCGTTTCACTATCCAGG	55.8

V	50	NGA76 F	GGAGAAAATGTCACTCTCCACC	60.1	231	>250	300

		NGA76 R	AGGCATGGGAGACATTTACG	57.8

V	61.1	ATHSO191 L	CTCCACCAATCATGCAAATG	55.8	148	156	146

		ATHSO191 R	TGATGTTGATGGAGATGGTCA	53.7

V	81.7	NGA129 F	TCAGGAGGAACTAAAGTGAGGG	60.1	177	179	172

		NGA129 R	CACACTGAAGATGGTCTTGAGG	60.1

[0181]
1 103 1 23 DNA Artificial sequence modified_base 11 I 1 ctggatccac nggnccnaay atg 23 2 23 DNA Artificial sequence modified_base 15 I 2 ctggatccrt artgngtnrc raa 23 3 24 DNA Artificial sequence MSH3 specific primer 636 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 3 tgctagtgcc tcttgcaagc tcat 24 4 27 DNA Artificial sequence Primer AP1 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia containing adapter sequences ligated to both its ends 4 ccatcctaat acgactcact atagggc 27 5 23 DNA Artificial sequence Primer AP2 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia containing adapter sequences ligated to both its ends 5 actcactata gggctcgagc ggc 23 6 30 DNA Artificial sequence MSH3 specific primer S525 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 6 aggttctgat tatgtgtgac gctttactta 30 7 29 DNA Artificial sequence MSH3 specific primer S51 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 7 ggatcgggta ctgggttttg agtgtgagg 29 8 24 DNA Artificial sequence MSH3 specific primer 635 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 8 gcacgtgctt gatggtgttt tcac 24 9 28 DNA Artificial sequence MSH3 specific primer S523 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 9 tcagacagta tccagcatgg cagaagta 28 10 33 DNA Artificial sequence MSH3 specific primer 1S5 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 10 atcccgggat gggcaagcaa aagcagcaga cga 33 11 27 DNA Artificial sequence MSH3 specific primer S53 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 11 gacaaagagc gaaatgaggc cccttgg 27 12 1250 DNA Arabidopsis thaliana ecotype Columbia Clone 52 12 cccgggatgg gcaagcaaaa gcagcagacg atttctcgtt tcttcgctcc caaacccaaa 60 tccccgactc acgaaccgaa tccggtagcc gaatcatcaa caccgccacc gaagatatcc 120 gccactgtat ccttctctcc ttccaagcgt aagcttctct ccgaccacct cgccgccgcg 180 tcacccaaaa agcctaaact ttctcctcac actcaaaacc cagtacccga tcccaattta 240 caccaaagat ttctccagag atttctggaa ccctcgccgg aggaatatgt tcccgaaacg 300 tcatcatcga ggaaatacac accattggaa cagcaagtgg tggagctaaa gagcaagtac 360 ccagatgtgg ttttgatggt ggaagttggt tacaggtaca gattcttcgg agaagacgcg 420 gagatcgcag cacgcgtgtt gggtatttac gctcatatgg atcacaattt catgacggcg 480 agtgtgccaa catttcgatt gaatttccat gtgagaagac tggtgaatgc aggatacaag 540 attggtgtag tgaagcagac tgaaactgca gccattaagt cccatggtgc aaaccggacc 600 ggcccttttt tccggggact gtcggcgttg tataccaaag ccacgcttga agcggctgag 660 gatataagtg gtggttgtgg tggtgaagaa ggttttggtt cacagagtaa tttcttggtt 720 tgtgttgtgg atgagagagt taagtcggag acattaggct gtggtattga aatgagtttt 780 gatgttagag tcggtgttgt tggcgttgaa atttcgacag gtgaagttgt ttatgaagag 840 ttcaatgata atttcatgag aagtggatta gaggctgtga ttttgagctt gtcaccagct 900 gagctgttgc ttggccagcc tctttcacaa caaactgaga agtttttggt ggcacatgct 960 ggacctacct caaacgttcg agtggaacgt gcctcactgg attgtttcag caatggtaat 1020 gcagtagatg aggttatttc attatgtgaa aaaatcagcg caggtaactt agaagatgat 1080 aaagaaatga agctggaggc tgctgaaaaa ggaatgtctt gcttgacagt tcatacaatt 1140 atgaacatgc cacatctgac tgttcaagcc ctcgccctaa cgttttgcca tctcaaacag 1200 tttggatttg aaaggatcct ttaccaaggg gcctcatttc gctctttg 1250 13 34 DNA Artificial sequence MSH3 specific primer 2S5 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 13 atcccgggtc aaaatgaaca agttggtttt agtc 34 14 27 DNA Artificial sequence MSH3 specific primer S52 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 14 gccacatctg actgttcaag ccctcgc 27 15 2110 DNA Arabidopsis thaliana ecotype Columbia Clone 13 15 gccacatctg actgttcaag ccctcgccct aacgttttgc catctcaaac agtttggatt 60 tgaaaggatc ctttaccaag gggcctcatt tcgctctttg tcaagtaaca cagagatgac 120 tctctcagcc aatactctgc aacagttgga ggttgtgaaa aataattcag atggatcgga 180 atctggctcc ttattccata atatgaatca cacacttaca gtatatggtt ccaggcttct 240 tagacactgg gtgactcatc ctctatgcga tagaaatttg atatctgctc ggcttgatgc 300 tgtttctgag atttctgctt gcatgggatc tcatagttct tcccagctca gcagtgagtt 360 ggttgaagaa ggttctgaga gagcaattgt atcacctgag ttttatctcg tgctctcctc 420 agtcttgaca gctatgtcta gatcatctga tattcaacgt ggaataacaa gaatctttca 480 tcggactgct aaagccacag agttcattgc agttatggaa gctattttac ttgcggggaa 540 gcaaattcag cggcttggca taaagcaaga ctctgaaatg aggagtatgc aatctgcaac 600 tgtgcgatct actcttttga gaaaattgat ttctgttatt tcatcccctg ttgtggttga 660 caatgccgga aaacttctct ctgccctaaa taaggaagcg gctgttcgag gtgacttgct 720 cgacatacta atcacttcca gcgaccaatt tcctgagctt gctgaagctc gccaagcagt 780 tttagtcatc agggaaaagc tggattcctc gatagcttca tttcgcaaga agctcgctat 840 tcgaaatttg gaatttcttc aagtgtcggg gatcacacat ttgatagagc tgcccgttga 900 ttccaaggtc cctatgaatt gggtgaaagt aaatagcacc aagaagacta ttcgatatca 960 tcccccagaa atagtagctg gcttggatga gctagctcta gcaactgaac atcttgccat 1020 tgtgaaccga gcttcgtggg atagtttcct caagagtttc agtagatact acacagattt 1080 taaggctgcc gttcaagctc ttgctgcact ggactgtttg cactcccttt caactctatc 1140 tagaaacaag aactatgtcc gtcccgagtt tgtggatgac tgtgaaccag ttgagataaa 1200 catacagtct ggtcgtcatc ctgtactgga gactatatta caagataact tcgtcccaaa 1260 tgacacaatt ttgcatgcag aaggggaata ttgccaaatt atcaccggac ctaacatggg 1320 aggaaagagc tgctatatcc gtcaagttgc tttaatttcc ataatggctc aggttggttc 1380 ctttgtacca gcgtcattcg ccaagctgca cgtgcttgat ggtgttttca ctcggatggg 1440 tgcttcagac agtatccagc atggcagaag tacctttcta gaagaattaa gtgaagcgtc 1500 acacataatc agaacctgtt cttctcgttc gcttgttata ttagatgagc ttggaagagg 1560 cactagcaca cacgacggtg tagccattgc ctatgcaaca ttacagcatc tcctagcaga 1620 aaagagatgt ttggttcttt ttgtcacgca ttaccctgaa atagctgaga tcagtaacgg 1680 attcccaggt tctgttggga cataccatgt ctcgtatctg acattgcaga aggataaagg 1740 cagttatgat catgatgatg tgacctacct atataagctt gtgcgtggtc tttgcagcag 1800 gagctttggt tttaaggttg ctcagcttgc ccagatacct ccatcatgta tacgtcgagc 1860 catttcaatg gctgcaaaat tggaagctga ggtacgtgca agagagagaa atacacgcat 1920 gggagaacca gaaggacatg aagaaccgag aggcgcagaa gaatctattt cggctctagg 1980 tgacttgttt gcagacctga aatttgctct ctctgaagag gacccttgga aagcattcga 2040 gtttttaaag catgcttgga agattgctgg caaaatcaga ctaaaaccaa cttgttcatt 2100 ttgacccggg 2110 16 29 DNA Artificial sequence MSH3 specific primer S51 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 16 ggatcgggta ctgggttttg agtgtgagg 29 17 30 DNA Artificial sequence MSH3 specific primer S525 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 17 aggttctgat tatgtgtgac gctttactta 30 18 3522 DNA Arabidopsis thaliana ecotype Columbia CDS (100)....(3342) AtMSH3 full-length cDNA and deduced sequence of the encoded polypeptide 18 cctaagaaag cgcgcgaaaa ttggcaaccc aagttcgcca tagccacgac cacgaccttc 60 catttctctt aaacggagga gattacgaat aaagcaatt 99 atg ggc aag caa aag cag cag acg att tct cgt ttc ttc gct ccc aaa 147 Met Gly Lys Gln Lys Gln Gln Thr Ile Ser Arg Phe Phe Ala Pro Lys 1 5 10 15 ccc aaa tcc ccg act cac gaa ccg aat ccg gta gcc gaa tca tca aca 195 Pro Lys Ser Pro Thr His Glu Pro Asn Pro Val Ala Glu Ser Ser Thr 20 25 30 ccg cca ccg aag ata tcc gcc act gta tcc ttc tct cct tcc aag cgt 243 Pro Pro Pro Lys Ile Ser Ala Thr Val Ser Phe Ser Pro Ser Lys Arg 35 40 45 aag ctt ctc tcc gac cac ctc gcc gcc gcg tca ccc aaa aag cct aaa 291 Lys Leu Leu Ser Asp His Leu Ala Ala Ala Ser Pro Lys Lys Pro Lys 50 55 60 ctt tct cct cac act caa aac cca gta ccc gat ccc aat tta cac caa 339 Leu Ser Pro His Thr Gln Asn Pro Val Pro Asp Pro Asn Leu His Gln 65 70 75 80 aga ttt ctc cag aga ttt ctg gaa ccc tcg ccg gag gaa tat gtt ccc 387 Arg Phe Leu Gln Arg Phe Leu Glu Pro Ser Pro Glu Glu Tyr Val Pro 85 90 95 gaa acg tca tca tcg agg aaa tac aca cca ttg gaa cag caa gtg gtg 435 Glu Thr Ser Ser Ser Arg Lys Tyr Thr Pro Leu Glu Gln Gln Val Val 100 105 110 gag cta aag agc aag tac cca gat gtg gtt ttg atg gtg gaa gtt ggt 483 Glu Leu Lys Ser Lys Tyr Pro Asp Val Val Leu Met Val Glu Val Gly 115 120 125 tac agg tac aga ttc ttc gga gaa gac gcg gag atc gca gca cgc gtg 531 Tyr Arg Tyr Arg Phe Phe Gly Glu Asp Ala Glu Ile Ala Ala Arg Val 130 135 140 ttg ggt att tac gct cat atg gat cac aat ttc atg acg gcg agt gtg 579 Leu Gly Ile Tyr Ala His Met Asp His Asn Phe Met Thr Ala Ser Val 145 150 155 160 cca aca ttt cga ttg aat ttc cat gtg aga aga ctg gtg aat gca gga 627 Pro Thr Phe Arg Leu Asn Phe His Val Arg Arg Leu Val Asn Ala Gly 165 170 175 tac aag att ggt gta gtg aag cag act gaa act gca gcc att aag tcc 675 Tyr Lys Ile Gly Val Val Lys Gln Thr Glu Thr Ala Ala Ile Lys Ser 180 185 190 cat ggt gca aac cgg acc ggc cct ttt ttc cgg gga ctg tcg gcg ttg 723 His Gly Ala Asn Arg Thr Gly Pro Phe Phe Arg Gly Leu Ser Ala Leu 195 200 205 tat acc aaa gcc acg ctt gaa gcg gct gag gat ata agt ggt ggt tgt 771 Tyr Thr Lys Ala Thr Leu Glu Ala Ala Glu Asp Ile Ser Gly Gly Cys 210 215 220 ggt ggt gaa gaa ggt ttt ggt tca cag agt aat ttc ttg gtt tgt gtt 819 Gly Gly Glu Glu Gly Phe Gly Ser Gln Ser Asn Phe Leu Val Cys Val 225 230 235 240 gtg gat gag aga gtt aag tcg gag aca tta ggc tgt ggt att gaa atg 867 Val Asp Glu Arg Val Lys Ser Glu Thr Leu Gly Cys Gly Ile Glu Met 245 250 255 agt ttt gat gtt aga gtc ggt gtt gtt ggc gtt gaa att tcg aca ggt 915 Ser Phe Asp Val Arg Val Gly Val Val Gly Val Glu Ile Ser Thr Gly 260 265 270 gaa gtt gtt tat gaa gag ttc aat gat aat ttc atg aga agt gga tta 963 Glu Val Val Tyr Glu Glu Phe Asn Asp Asn Phe Met Arg Ser Gly Leu 275 280 285 gag gct gtg att ttg agc ttg tca cca gct gag ctg ttg ctt ggc cag 1011 Glu Ala Val Ile Leu Ser Leu Ser Pro Ala Glu Leu Leu Leu Gly Gln 290 295 300 cct ctt tca caa caa act gag aag ttt ttg gtg gca cat gct gga cct 1059 Pro Leu Ser Gln Gln Thr Glu Lys Phe Leu Val Ala Met Ala Gly Pro 305 310 315 320 acc tca aac gtt cga gtg gaa cgt gcc tca ctg gat tgt ttc agc aat 1107 Thr Ser Asn Val Arg Val Glu Arg Ala Ser Leu Asp Cys Phe Ser Asn 325 330 335 ggt aat gca gta gat gag gtt att tca tta tgt gaa aaa atc agc gca 1155 Gly Asn Ala Val Asp Glu Val Ile Ser Leu Cys Glu Lys Ile Ser Ala 340 345 350 ggt aac tta gaa gat gat aaa gaa atg aag ctg gag gct gct gaa aaa 1203 Gly Asn Leu Glu Asp Asp Lys Glu Met Lys Leu Glu Ala Ala Glu Lys 355 360 365 gga atg tct tgc ttg aca gtt cat aca att atg aac atg cca cat ctg 1251 Gly Met Ser Cys Leu Thr Val His Thr Ile Met Asn Met Pro His Leu 370 375 380 act gtt caa gcc ctc gcc cta acg ttt tgc cat ctc aaa cag ttt gga 1299 Thr Val Gln Ala Leu Ala Leu Thr Phe Cys His Leu Lys Gln Phe Gly 385 390 395 400 ttt gaa agg atc ctt tac caa ggg gcc tca ttt cgc tct ttg tca agt 1347 Phe Glu Arg Ile Leu Tyr Gln Gly Ala Ser Phe Arg Ser Leu Ser Ser 405 410 415 aac aca gag atg act ctc tca gcc aat act ctg caa cag ttg gag gtt 1395 Asn Thr Glu Met Thr Leu Ser Ala Asn Thr Leu Gln Gln Leu Glu Val 420 425 430 gtg aaa aat aat tca gat gga tcg gaa tct ggc tcc tta ttc cat aat 1443 Val Lys Asn Asn Ser Asp Gly Ser Glu Ser Gly Ser Leu Phe His Asn 435 440 445 atg aat cac aca ctt aca gta tat gct tcc agg ctt ctt aga cac tgg 1491 Met Asn His Thr Leu Thr Val Tyr Gly Ser Arg Leu Leu Arg His Trp 450 455 460 gtg act cat cct cta tgc gat aga aat ttg ata tct gct cgg ctt gat 1539 Val Thr His Pro Leu Cys Asp Arg Asn Leu Ile Ser Ala Arg Leu Asp 465 470 475 480 gct gtt tct gag att tct gct tgc atg gga tct cat agt tct tcc cag 1587 Ala Val Ser Glu Ile Ser Ala Cys Met Gly Ser His Ser Ser Ser Gln 485 490 495 ctc agc agt gag ttg gtt gaa gaa ggt tct gag aga gca att gta tca 1635 Leu Ser Ser Glu Leu Val Glu Glu Gly Ser Glu Arg Ala Ile Val Ser 500 505 510 cct gag ttt tat ctc gtg ctc tcc tca gtc ttg aca gct atg tct aga 1683 Pro Glu Phe Tyr Leu Val Leu Ser Ser Val Leu Thr Ala Met Ser Arg 515 520 525 tca tct gat att caa cgt gga ata aca aga atc ttt cat cgg act gct 1731 Ser Ser Asp Ile Gln Arg Gly Ile Thr Arg Ile Phe His Arg Thr Ala 530 535 540 aaa gcc aca gag ttc att gca gtt atg gaa gct att tta ctt gcg ggg 1779 Lys Ala Thr Glu Phe Ile Ala Val Met Glu Ala Ile Leu Leu Ala Gly 545 550 555 560 aag caa att cag cgg ctt ggc ata aag caa gac tct gaa atg agg agt 1827 Lys Gln Ile Gln Arg Leu Gly Ile Lys Gln Asp Ser Glu Met Arg Ser 565 570 575 atg caa tct gca act gtg cga tct act ctt ttg aga aaa ttg att tct 1875 Met Gln Ser Ala Thr Val Arg Ser Thr Leu Leu Arg Lys Leu Ile Ser 580 585 590 gtt att tca tcc cct gtt gtg gtt gac aat gcc gga aaa ctt ctc tct 1923 Val Ile Ser Ser Pro Val Val Val Asp Asn Ala Gly Lys Leu Leu Ser 595 600 605 gcc cta aat aag gaa gcg gct gtt cga ggt gac ttg ctc gac ata cta 1971 Ala Leu Asn Lys Glu Ala Ala Val Arg Gly Asp Leu Leu Asp Ile Leu 610 615 620 atc act tcc agc gac caa ttt cct gag ctt gct gaa gct cgc caa gca 2019 Ile Thr Ser Ser Asp Gln Phe Pro Glu Leu Ala Glu Ala Arg Gln Ala 625 630 635 640 gtt tta gtc atc agg gaa aag ctg gat tcc tcg ata gct tca ttt cgc 2067 Val Leu Val Ile Arg Glu Lys Leu Asp Ser Ser Ile Ala Ser Phe Arg 645 650 655 aag aag ctc gct att cga aat ttg gaa ttt ctt caa gtg tcg ggg atc 2115 Lys Lys Leu Ala Ile Arg Asn Leu Glu Phe Leu Gln Val Ser Gly Ile 660 665 670 aca cat ttg ata gag ctg ccc gtt gat tcc aag gtc cct atg aat tgg 2163 Thr His Leu Ile Glu Leu Pro Val Asp Ser Lys Val Pro His Asn Trp 675 680 685 gtg aaa gta aat agc acc aag aag act att cga tat cat ccc cca gaa 2211 Val Lys Val Asn Ser Thr Lys Lys Thr Ile Arg Tyr His Pro Pro Glu 690 695 700 ata gta gct ggc ttg gat gag cta gct cta gca act gaa cat ctt gcc 2259 Ile Val Ala Gly Leu Asp Glu Leu Ala Leu Ala Thr Glu His Leu Ala 705 710 715 720 att gtg aac cga gct tcg tgg gat agt ttc ctc aag agt ttc agt aga 2307 Ile Val Asn Arg Ala Ser Trp Asp Ser Phe Leu Lys Ser Phe Ser Arg 725 730 735 tac tac aca gat ttt aag gct gcc gtt caa gct ctt gct gca ctg gac 2355 Tyr Tyr Thr Asp Phe Lys Ala Ala Val Gln Ala Leu Ala Ala Leu Asp 740 745 750 tgt ttg cac tcc ctt tca act cta tct aga aac aag aac tat gtc cgt 2403 Cys Leu His Ser Leu Ser Thr Leu Ser Arg Asn Lys Asn Tyr Val Arg 755 760 765 ccc gag ttt gtg gat gac tgt gaa cca gtt gag ata aac ata cag tct 2451 Pro Glu Phe Val Asp Asp Cys Glu Pro Val Glu Ile Asn Ile Gln Ser 770 775 780 ggt cgt cat cct gta ctg gag act ata tta caa gat aac ttc gtc cca 2499 Gly Arg His Pro Val Leu Glu Thr Ile Leu Gln Asp Asn Phe Val Pro 785 790 795 800 aat gac aca att ttg cat gca gaa ggg gaa tat tgc caa att atc acc 2547 Asn Asp Thr Ile Leu His Ala Glu Gly Glu Tyr Cys Gln Ile Ile Thr 805 810 815 gga cct aac atg gga gga aag agc tgc tat atc cgt caa gtt gct tta 2595 Gly Pro Asn Met Gly Gly Lys Ser Cys Tyr Ile Arg Gln Val Ala Leu 820 825 830 att tcc ata atg gct cag gtt ggt tcc ttt gta cca gcg tca ttc gcc 2643 Ile Ser Ile Met Ala Gln Val Gly Ser Phe Val Pro Ala Ser Phe Ala 835 840 845 aag ctg cac gtg ctt gat ggt gtt ttc act cgg atg ggt gct tca gac 2691 Lys Leu His Val Leu Asp Gly Val Phe Thr Arg Met Gly Ala Ser Asp 850 855 860 agt atc cag cat ggc aga agt acc ttt cta gaa gaa tta agt gaa gcg 2739 Ser Ile Gln His Gly Arg Ser Thr Phe Leu Glu Glu Leu Ser Glu Ala 865 870 875 880 tca cac ata atc aga acc tgt tct tct cgt tcg ctt gtt ata tta gat 2787 Ser His Ile Ile Arg Thr Cys Ser Ser Arg Ser Leu Val Ile Leu Asp 885 890 895 gag ctt gga aga ggc act agc aca cac gac ggt gta gcc att gcc tat 2835 Glu Leu Gly Arg Gly Thr Ser Thr His Asp Gly Val Ala Ile Ala Tyr 900 905 910 gca aca tta cag cat ctc cta gca gaa aag aga tgt ttg gtt ctt ttt 2883 Ala Thr Leu Gln His Leu Leu Ala Glu Lys Arg Cys Leu Val Leu Phe 915 920 925 gtc acg cat tac cct gaa ata gct gag atc agt aac gga ttc cca ggt 2931 Val Thr His Tyr Pro Glu Ile Ala Glu Ile Ser Asn Gly Phe Pro Gly 930 935 940 tct gtt ggg aca tac cat gtc tcg tat ctg aca ttg cag aag gat aaa 2979 Ser Val Gly Thr Tyr His Val Ser Tyr Leu Thr Leu Gln Lys Asp Lys 945 950 955 960 ggc agt tat gat cat gat gat gtg acc tac cta tat aag ctt gtg cgt 3027 Gly Ser Tyr Asp His Asp Asp Val Thr Tyr Leu Tyr Lys Leu Val Arg 965 970 975 ggt ctt tgc agc agg agc ttt ggt ttt aag gtt gct cag ctt gcc cag 3075 Gly Leu Cys Ser Arg Ser Phe Gly Phe Lys Val Ala Gln Leu Ala Gln 980 985 990 ata cct cca tca tgt ata cgt cga gcc att tca atg gct gca aaa ttg 3123 Ile Pro Pro Ser Cys Ile Arg Arg Ala Ile Ser Met Ala Ala Lys Leu 995 1000 1005 gaa gct gag gta cgt gca aga gag aga aat aca cgc atg gga gaa cca 3171 Glu Ala Glu Val Arg Ala Arg Glu Arg Asn Thr Arg Met Gly Glu Pro 1010 1015 1020 gaa gga cat gaa gaa ccg aga ggc gca gaa gaa tct att tcg gct cta 3219 Glu Gly His Glu Glu Pro Arg Gly Ala Glu Glu Ser Ile Ser Ala Leu 1025 1030 1035 1040 ggt gac ttg ttt gca gac ctg aaa ttt gct ctc tct gaa gag gac cct 3267 Gly Asp Leu Phe Ala Asp Leu Lys Phe Ala Leu Ser Glu Glu Asp Pro 1045 1050 1055 tgg aaa gca ttc gag ttt tta aag cat gct tgg aag att gct ggc aaa 3315 Trp Lys Ala Phe Glu Phe Leu Lys His Ala Trp Lys Ile Ala Gly Lys 1060 1065 1070 atc aga cta aaa cca act tgt tca ttt tgatttaatc ttaacattat 3362 Ile Arg Leu Lys Pro Thr Cys Ser Phe 1075 1080 agcaactgca aggtcttgat catctgttag ttgcgtacta acttatgtgt attagtataa 3422 caagaaaaga gaattagaga gatggattct aatccggtgt tgcagtacat cttttctcca 3482 cccgcataaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3522 19 1081 PRT Arabidopsis thaliana ecotype Columbia Polypeptide MSH3 19 Met Gly Lys Gln Lys Gln Gln Thr Ile Ser Arg Phe Phe Ala Pro Lys 1 5 10 15 Pro Lys Ser Pro Thr His Glu Pro Asn Pro Val Ala Glu Ser Ser Thr 20 25 30 Pro Pro Pro Lys Ile Ser Ala Thr Val Ser Phe Ser Pro Ser Lys Arg 35 40 45 Lys Leu Leu Ser Asp His Leu Ala Ala Ala Ser Pro Lys Lys Pro Lys 50 55 60 Leu Ser Pro His Thr Gln Asn Pro Val Pro Asp Pro Asn Leu His Gln 65 70 75 80 Arg Phe Leu Gln Arg Phe Leu Glu Pro Ser Pro Glu Glu Tyr Val Pro 85 90 95 Glu Thr Ser Ser Ser Arg Lys Tyr Thr Pro Leu Glu Gln Gln Val Val 100 105 110 Glu Leu Lys Ser Lys Tyr Pro Asp Val Val Leu Met Val Glu Val Gly 115 120 125 Tyr Arg Tyr Arg Phe Phe Gly Glu Asp Ala Glu Ile Ala Ala Arg Val 130 135 140 Leu Gly Ile Tyr Ala His Met Asp His Asn Phe Met Thr Ala Ser Val 145 150 155 160 Pro Thr Phe Arg Leu Asn Phe His Val Arg Arg Leu Val Asn Ala Gly 165 170 175 Tyr Lys Ile Gly Val Val Lys Gln Thr Glu Thr Ala Ala Ile Lys Ser 180 185 190 His Gly Ala Asn Arg Thr Gly Pro Phe Phe Arg Gly Leu Ser Ala Leu 195 200 205 Tyr Thr Lys Ala Thr Leu Glu Ala Ala Glu Asp Ile Ser Gly Gly Cys 210 215 220 Gly Gly Glu Glu Gly Phe Gly Ser Gln Ser Asn Phe Leu Val Cys Val 225 230 235 240 Val Asp Glu Arg Val Lys Ser Glu Thr Leu Gly Cys Gly Ile Glu Met 245 250 255 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 Glu Leu Leu Leu Gly Gln 290 295 300 Pro Leu Ser Gln Gln Thr Glu Lys Phe Leu Val Ala Met Ala Gly Pro 305 310 315 320 Thr Ser Asn Val Arg Val Glu Arg Ala Ser Leu Asp Cys Phe Ser Asn 325 330 335 Gly Asn Ala Val Asp Glu Val Ile Ser Leu Cys Glu Lys Ile Ser Ala 340 345 350 Gly Asn Leu Glu Asp Asp Lys Glu Met Lys Leu Glu Ala Ala Glu Lys 355 360 365 Gly Met Ser Cys Leu Thr Val His Thr Ile Met Asn Met Pro His Leu 370 375 380 Thr Val Gln Ala Leu Ala Leu Thr Phe Cys His Leu Lys Gln Phe Gly 385 390 395 400 Phe Glu Arg Ile Leu Tyr Gln Gly Ala Ser Phe Arg Ser Leu Ser Ser 405 410 415 Asn Thr Glu Met Thr Leu Ser Ala Asn Thr Leu Gln Gln Leu Glu Val 420 425 430 Val Lys Asn Asn Ser Asp Gly Ser Glu Ser Gly Ser Leu Phe His Asn 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 Ile Ser Ala Arg Leu Asp 465 470 475 480 Ala Val Ser Glu Ile Ser Ala Cys Met Gly Ser His Ser Ser Ser Gln 485 490 495 Leu Ser Ser Glu Leu Val Glu Glu Gly Ser Glu Arg Ala Ile Val Ser 500 505 510 Pro Glu Phe Tyr Leu Val Leu Ser Ser Val Leu Thr Ala Met Ser Arg 515 520 525 Ser Ser Asp Ile Gln Arg Gly Ile Thr Arg Ile Phe His Arg Thr Ala 530 535 540 Lys Ala Thr Glu Phe Ile Ala Val Met Glu Ala Ile Leu Leu Ala Gly 545 550 555 560 Lys Gln Ile Gln Arg Leu Gly Ile Lys Gln Asp Ser Glu Met Arg Ser 565 570 575 Met Gln Ser Ala Thr Val Arg Ser Thr Leu Leu Arg Lys Leu Ile Ser 580 585 590 Val Ile Ser Ser Pro Val Val Val Asp Asn Ala Gly Lys Leu Leu Ser 595 600 605 Ala Leu Asn Lys Glu Ala Ala Val Arg Gly Asp Leu Leu Asp Ile Leu 610 615 620 Ile Thr Ser Ser Asp Gln Phe Pro Glu Leu Ala Glu Ala Arg Gln Ala 625 630 635 640 Val Leu Val Ile Arg Glu Lys Leu Asp Ser Ser Ile Ala Ser Phe Arg 645 650 655 Lys Lys Leu Ala Ile Arg Asn Leu Glu Phe Leu Gln Val Ser Gly Ile 660 665 670 Thr His Leu Ile Glu Leu Pro Val Asp Ser Lys Val Pro His Asn Trp 675 680 685 Val Lys Val Asn Ser Thr Lys Lys Thr Ile Arg Tyr His Pro Pro Glu 690 695 700 Ile Val Ala Gly Leu Asp Glu Leu Ala Leu Ala Thr Glu His Leu Ala 705 710 715 720 Ile Val Asn Arg Ala Ser Trp Asp Ser Phe Leu Lys Ser Phe Ser Arg 725 730 735 Tyr Tyr Thr Asp Phe Lys Ala Ala Val Gln Ala Leu Ala Ala Leu Asp 740 745 750 Cys Leu His Ser Leu Ser Thr Leu Ser Arg Asn Lys Asn Tyr Val Arg 755 760 765 Pro Glu Phe Val Asp Asp Cys Glu Pro Val Glu Ile Asn Ile Gln Ser 770 775 780 Gly Arg His Pro Val Leu Glu Thr Ile Leu Gln Asp Asn Phe Val Pro 785 790 795 800 Asn Asp Thr Ile Leu His Ala Glu Gly Glu Tyr Cys Gln Ile Ile Thr 805 810 815 Gly Pro Asn Met Gly Gly Lys Ser Cys Tyr Ile Arg Gln Val Ala Leu 820 825 830 Ile Ser Ile Met Ala Gln Val Gly Ser Phe Val Pro Ala Ser Phe Ala 835 840 845 Lys Leu His Val Leu Asp Gly Val Phe Thr Arg Met Gly Ala Ser Asp 850 855 860 Ser Ile Gln His Gly Arg Ser Thr Phe Leu Glu Glu Leu Ser Glu Ala 865 870 875 880 Ser His Ile Ile Arg Thr Cys Ser Ser Arg Ser Leu Val Ile Leu Asp 885 890 895 Glu Leu Gly Arg Gly Thr Ser Thr His Asp Gly Val Ala Ile Ala Tyr 900 905 910 Ala Thr Leu Gln His Leu Leu Ala Glu Lys Arg Cys Leu Val Leu Phe 915 920 925 Val Thr His Tyr Pro Glu Ile Ala Glu Ile Ser Asn Gly Phe Pro Gly 930 935 940 Ser Val Gly Thr Tyr His Val Ser Tyr Leu Thr Leu Gln Lys Asp Lys 945 950 955 960 Gly Ser Tyr Asp His Asp Asp Val Thr Tyr Leu Tyr Lys Leu Val Arg 965 970 975 Gly Leu Cys Ser Arg Ser Phe Gly Phe Lys Val Ala Gln Leu Ala Gln 980 985 990 Ile Pro Pro Ser Cys Ile Arg Arg Ala Ile Ser Met Ala Ala Lys Leu 995 1000 1005 Glu Ala Glu Val Arg Ala Arg Glu Arg Asn Thr Arg Met Gly Glu Pro 1010 1015 1020 Glu Gly His Glu Glu Pro Arg Gly Ala Glu Glu Ser Ile Ser Ala Leu 1025 1030 1035 1040 Gly Asp Leu Phe Ala Asp Leu Lys Phe Ala Leu Ser Glu Glu Asp Pro 1045 1050 1055 Trp Lys Ala Phe Glu Phe Leu Lys His Ala Trp Lys Ile Ala Gly Lys 1060 1065 1070 Ile Arg Leu Lys Pro Thr Cys Ser Phe 1075 1080 20 24 DNA Artificial sequence MSH6 specific primer 638 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 20 tctctaccag gtgacgaaaa accg 24 21 28 DNA Artificial sequence Primer S81 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 21 cgtcgccttt agcatcccct tccttcac 28 22 30 DNA Artificial sequence MSH6 specific primer S823 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 22 gcttggcgca tctaatagaa tcatgacagg 30 23 24 DNA Artificial sequence MSH6 specific primer 637 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 23 gacagcgtca gttcttcaga atgc 24 24 33 DNA Artificial sequence MSH6 specific primer 1S8 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 24 atcccgggat gcagcgccag agatcgattt tgt 33 25 27 DNA Artificial sequence MSH6 specific primer S83 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 25 cgctatctat ggctgcttcg aattgag 27 26 2188 DNA Arabidopsis thaliana ecotype Columbia Clone 43 26 cccgggatgc agcgccagag atcgattttg tctttcttcc aaaaacccac ggcggcgact 60 acgaagggtt tggtttccgg cgatgctgct agcggcgggg gcggcagcgg aggaccacga 120 tttaatgtga aggaagggga tgctaaaggc gacgcttctg tacgttttgc tgtttcgaaa 180 tctgtcgatg aggttagagg aacggatact ccaccggaga aggttccgcg tcgtgtcctg 240 ccgtctggat ttaagccggc tgaatccgcc ggtgatgctt cgtccctgtt ctccaatatt 300 atgcataagt ttgtaaaagt cgatgatcga gattgttctg gagagaggag ccgagaagat 360 gttgttccgc tgaatgattc atctctatgt atgaaggcta atgatgttat tcctcaattt 420 cgttccaata atggtaaaac tcaagaaaga aaccatgctt ttagtttcag tgggagagct 480 gaacttagat cagtagaaga tataggagta gatggcgatg ttcctggtcc agaaacacca 540 gggatgcgtc cacgtgcttc tcgcttgaag cgagttctgg aggatgaaat gacttttaag 600 gaggataagg ttcctgtatt ggactctaac aaaaggctga aaatgctcca ggatccggtt 660 tgtggagaga agaaagaagt aaacgaagga accaaatttg aatggcttga gtcttctcga 720 atcagggatg ccaatagaag acgtcctgat gatccccttt acgatagaaa gaccttacac 780 ataccacctg atgttttcaa gaaaatgtct gcatcacaaa agcaatattg gagtgttaag 840 agtgaatata tggacattgt gcttttcttt aaagtgggga aattttatga gctgtatgag 900 ctagatgcgg aattaggtca caaggagctt gactggaaga tgaccatgag tggtgtggga 960 aaatgcagac aggttggtat ctctgaaagt gggatagatg aggcagtgca aaagctatta 1020 gctcgtggat ataaagttgg acgaatcgag cagctagaaa catctgacca agcaaaagcc 1080 agaggtgcta atactataat tccaaggaag ctagttcagg tattaactcc atcaacagca 1140 agcgagggaa acatcgggcc tgatgccgtc catcttcttg ctataaaaga gatcaaaatg 1200 gagctacaaa agtgttcaac tgtgtatgga tttgcttttg ttgactgtgc tgccttgagg 1260 ttttgggttg ggtccatcag cgatgatgca tcatgtgctg ctcttggagc gttattgatg 1320 caggtttctc caaaggaagt gttatatgac agtaaagggc tatcaagaga agcacaaaag 1380 gctctaagga aatatacgtt gacagggtct acggcggtac agttggctcc agtaccacaa 1440 gtaatggggg atacagatgc tgctggagtt agaaatataa tagaatctaa cggatacttt 1500 aaaggttctt ctgaatcatg gaactgtgct gttgatggtc taaatgaatg tgatgttgcc 1560 cttagtgctc ttggagagct aattaatcat ctgtctaggc taaagctaga agatgtactt 1620 aagcatgggg atatttttcc ataccaagtt tacaggggtt gtctcagaat tgatggccag 1680 acgatggtaa atcttgagat atttaacaat agctgtgatg gtggtccttc agggaccttg 1740 tacaaatatc ttgataactg tgttagtcca actggtaagc gactcttaag gaattggatc 1800 tgccatccac tcaaagatgt agaaagcatc aataaacggc ttgatgtagt tgaagaattc 1860 acggcaaact cagaaagtat gcaaatcact ggccagtatc tccacaaact tccagactta 1920 gaaagactgc tcggacgcat caagtctagc gttcgatcat cagcctctgt gttgcctgct 1980 cttctgggga aaaaagtgct gaaacaacga gttaaagcat ttgggcaaat tgtgaaaggg 2040 ttcagaagtg gaattgatct gttgttggct ctacagaagg aatcaaatat gatgagtttg 2100 ctttataaac tctgtaaact tcctatatta gtaggaaaaa gcgggctaga gttatttctt 2160 tctcaattcg aagcagccat agatagcg 2188 27 1385 DNA Arabidopsis thaliana ecotype Columbia Clone 62 27 catcagcctc tgtgttgcct gctcttctgg ggaaaaaagt gctgaaacaa cgagttaaag 60 catttgggca aattgtgaaa gggttcagaa gtggaattga tctgttgttg gctctacaga 120 aggaatcaaa tatgatgagt ttgctttata aactctgtaa acttcctata ttagtaggaa 180 aaagcgggct agagttattt ctttctcaat tcgaagcagc catagatagc gactttccaa 240 attatcagaa ccaagatgtg acagatgaaa acgctgaaac tctcacaata cttatcgaac 300 tttttatcga aagagcaact caatggtctg aggtcattca caccataagc tgcctagatg 360 tcctgagatc ttttgcaatc gcagcaagtc tctctgctgg aagcatggcc aggcctgtta 420 tttttcccga atcagaagct acagatcaga atcagaaaac aaaagggcca atacttaaaa 480 tccaaggact atggcatcca tttgcagttg cagccgatgg tcaattgcct gttccgaatg 540 atatactcct tggcgaggct agaagaagca gtggcagcat tcatcctcgg tcattgttac 600 tgacgggacc aaacatgggc ggaaaatcaa ctcttcttcg tgcaacatgt ctggccgtta 660 tctttgccca acttggctgc tacgtgccgt gtgagtcttg cgaaatctcc ctcgtggata 720 ctatcttcac aaggcttggc gcatctgata gaatcatgac aggagagagt acctttttgg 780 tagaatgcac tgagacagcg tcagttcttc agaatgcaac tcaggattca ctagtaatcc 840 ttgacgaact gggcagagga actagtactt tcgatggata cgccattgca tactcggttt 900 ttcgtcacct ggtagagaaa gttcaatgtc ggatgctctt tgcaacacat taccaccctc 960 tcaccaagga attcgcgtct cacccacgtg tcacctcgaa acacatggct tgcgcattca 1020 aatcaagatc tgattatcaa ccacgtggtt gtgatcaaga cctagtgttc ttgtaccgtt 1080 taaccgaggg agcttgtcct gagagctacg gacttcaagt ggcactcatg gctggaatac 1140 caaaccaagt ggttgaaaca gcatcaggtg ctgctcaagc catgaagaga tcaattgggg 1200 aaaacttcaa gtcaagtgag ctaagatctg agttctcaag tctgcatgaa gactggctca 1260 agtcattggt gggtatttct cgagtcgccc acaacaatgc ccccattggc gaagatgact 1320 acgacacttt gttttgctta tggcatgaga tcaaatcctc ttactgtgtt cccaaataac 1380 ccggg 1385 28 34 DNA Artificial sequence MSH6 specific primer 2S8 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 28 atcccgggtt atttgggaac acagtaagag gatt 34 29 27 DNA Artificial sequence MSH6 specific primer S82 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 29 gcgttcgatc atcagcctct gtgttgc 27 30 3606 DNA Arabidopsis thaliana ecotype Columbia CDS (142)....(3468) AtMSH6 full-length cDNA and deduced sequence of the encoded polypeptide 30 aaaagttgag ccctgaggag tatcgtttcc gccatttcta cgacgcaagg cgaaaatttt 60 tggcgccaat ctttcccccc tttcgaattc tctcagctca aaacatcgtt tctctctcac 120 tctctctcac aattccaaaa a atg cag cgc cag aga tcg att ttg tct ttc 171 Met Gln Arg Gln Arg Ser Ile Leu Ser Phe 1 5 10 ttc caa aaa ccc acc gcg gcg act acg aag ggt ttg gtt tcc ggc gat 219 Phe Gln Lys Pro Thr Ala Ala Thr Thr Lys Gly Leu Val Ser Gly Asp 15 20 25 gct gct agc ggc ggg ggc ggc agc gga gga cca cga ttt aat gtg aag 267 Ala Ala Ser Gly Gly Gly Gly Ser Gly Gly Pro Arg Phe Asn Val Arg 30 35 40 gaa ggg gat gct aaa ggc gac gct tct gta cgt ttt gct gtt tcg aaa 315 Glu Gly Asp Ala Lys Gly Asp Ala Ser Val Arg Phe Ala Val Ser Lys 45 50 55 tct gtc gat gag gtt aga gga acg gat act cca ccg gag aag gtt ccg 363 Ser Val Asp Glu Val Arg Gly Thr Asp Thr Pro Pro Glu Lys Val Pro 60 65 70 cgt cgt gtc ctg ccg tct gga ttt aag ccg gct gaa tcc gcc gst gat 411 Arg Arg Val Leu Pro Ser Gly Phe Lys Pro Ala Glu Ser Ala Gly Asp 75 80 85 90 gct tcg tcc ctg ttc tcc aat att atg cat aag ttt gta aaa gtc gat 459 Ala Ser Ser Leu Phe Ser Asn Ile Met His Lys Phe Val Lys Val Asp 95 100 105 gat cga gat tgt tct gga gag agg agc cga gaa gat gtt gtt ccg ctg 507 Asp Arg Asp Cys Ser Gly Glu Arg Ser Arg Glu Asp Val Val Pro Leu 110 115 120 aat gat tca tct cta tgt atg aag gct aat gat gtt att cct caa ttt 555 Asn Asp Ser Ser Leu Cys Met Lys Ala Asn Asp Val Ile Pro Gln Phe 125 130 135 cgt tcc aat aat ggt aaa act caa gaa aga aac cat gct ttt agt ttc 603 Arg Ser Asn Asn Gly Lys Thr Gln Glu Arg Asn His Ala Phe Ser Phe 140 145 150 agt ggg aga gct gaa ctt aga tca gta gaa gat ata gga gta gat ggc 651 Ser Gly Arg Ala Glu Leu Arg Ser Val Glu Asp Ile Gly Val Asp Gly 155 160 165 170 gat gtt cct ggt cca gaa aca cca ggg atg cgt cca cgt gct tct cgc 699 Asp Val Pro Gly Pro Glu Thr Pro Gly Met Arg Pro Arg Ala Ser Arg 175 180 185 ttg aag cga gtt ctg gag gat gaa atg act ttt aag gag gat aag gtt 747 Leu Lys Arg Val Leu Glu Asp Glu Met Thr Phe Lys Glu Asp Lys Val 190 195 200 cct gta ttg gac tct aac aaa agg ctg aaa atg ctc cag gat ccg gtt 795 Pro Val Leu Asp Ser Asn Lys Arg Leu Lys Met Leu Gln Asp Pro Val 205 210 215 tgt gga gag aag aaa gaa gta aac gaa gga acc aaa ttt gaa tgg ctt 843 Cys Gly Glu Lys Lys Glu Val Asn Glu Gly Thr Lys Phe Glu Trp Leu 220 225 230 gag tct tct cga atc agg gat gcc aat aga aga cgt cct gat gat ccc 891 Glu Ser Ser Arg Ile Arg Asp Ala Asn Arg Arg Arg Pro Asp Asp Pro 235 240 245 250 ctt tac gat aga aag acc tta cac ata cca cct gat gtt ttc aag aaa 939 Leu Tyr Asp Arg Lys Thr Leu His Ile Pro Pro Asp Val Phe Lys Lys 255 260 265 atg tct gca tca caa aag caa tat tgg agt gtt aag agt gaa tat atg 987 Met Ser Ala Ser Gln Lys Gln Tyr Trp Ser Val Lys Ser Glu Tyr Met 270 275 280 gac att gtg ctt ttc ttt aaa gtg ggg aaa ttt tat gag ctg tat gag 1035 Asp Ile Val Leu Phe Phe Lys Val Gly Lys Phe Tyr Glu Leu Tyr Glu 285 290 295 cta gat gcg gaa tta ggt cac aag gag ctt gac tgg aag atg acc atg 1083 Leu Asp Ala Glu Leu Gly His Lys Glu Leu Asp Trp Lys Met Thr Met 300 305 310 agt ggt gtg gga aaa tgc aga cag gtt ggt atc tct gaa agt ggg ata 1131 Ser Gly Val Gly Lys Cys Arg Gln Val Gly Ile Ser Glu Ser Gly Ile 315 320 325 330 gat gag gca gtg caa aag cta tta gct cgt gga tat aaa gtt gga cga 1179 Asp Glu Ala Val Gln Lys Leu Leu Ala Arg Gly Tyr Lys Val Gly Arg 335 340 345 atc gag cag cta gaa aca tct gac caa gca aaa gcc aga ggt gct aat 1227 Ile Glu Gln Leu Glu Thr Ser Asp Gln Ala Lys Ala Arg Gly Ala Asn 350 355 360 act ata att cca agg aag cta gtt cag gta tta act cca tca aca gca 1275 Thr Ile Ile Pro Arg Lys Leu Val Gln Val Leu Thr Pro Ser Thr Ala 365 370 375 agc gag gga aac atc ggg cct gat gcc gtc cat ctt ctt gct ata aaa 1323 Ser Glu Gly Asn Ile Gly Pro Asp Ala Val His Leu Leu Ala Ile Lys 380 385 390 gag atc aaa atg gag cta caa aag tgt tca act gtg tat gga ttt gct 1371 Glu Ile Lys Met Glu Leu Gln Lys Cys Ser Thr Val Tyr Gly Phe Ala 395 400 405 410 ttt gtt gac tgt gct gcc ttg agg ttt tgg gtt ggg tcc atc agc gat 1419 Phe Val Asp Cys Ala Ala Leu Arg Phe Trp Val Gly Ser Ile Ser Asp 415 420 425 gat gca tca tgt gct gct ctt gga gcg tta ttg atg cag gtt tct cca 1467 Asp Ala Ser Cys Ala Ala Leu Gly Ala Leu Leu Met Gln Val Ser Pro 430 435 440 aag gaa gtg tta tat gac agt aaa ggg cta tca aga gaa gca caa aag 1515 Lys Glu Val Leu Tyr Asp Ser Lys Gly Leu Ser Arg Glu Ala Gln Lys 445 450 455 gct cta agg aaa tat acg ttg aca ggg tct acg gcg gta cag ttg gct 1563 Ala Leu Arg Lys Tyr Thr Leu Thr Gly Ser Thr Ala Val Gln Leu Ala 460 465 470 cca gta cca caa gta atg ggg gat aca gat gct gct gga gtt aga aat 1611 Pro Val Pro Gln Val Met Gly Asp Thr Asp Ala Ala Gly Val Arg Asn 475 480 485 490 ata ata gaa tct aac gga tac ttt aaa ggt tct tct gaa tca tgg aac 1659 Ile Ile Glu Ser Asn Gly Tyr Phe Lys Gly Ser Ser Glu Ser Trp Asn 495 500 505 tgt gct gtt gat ggt cta aat gaa tgt gat gtt gcc ctt agt gct ctt 1707 Cys Ala Val Asp Gly Leu Asn Glu Cys Asp Val Ala Leu Ser Ala Leu 510 515 520 gga gag cta att aat cat ctg tct agg cta aag cta gaa gat gta ctt 1755 Gly Glu Leu Ile Asn His Leu Ser Arg Leu Lys Leu Glu Asp Val Leu 525 530 535 aag cat ggg gat att ttt cca tac caa gtt tac agg ggt tgt ctc aga 1803 Lys His Gly Asp Ile Phe Pro Tyr Gln Val Tyr Arg Gly Cys Leu Arg 540 545 550 att gat ggc cag acg atg gta aat ctt gag ata ttt aac aat agc tgt 1851 Ile Asp Gly Gln Thr Met Val Asn Leu Glu Ile Phe Asn Asn Ser Cys 555 560 565 570 gat ggt ggt cct tca ggg acc ttg tac aaa tat ctt gat aac tgt gtt 1899 Asp Gly Gly Pro Ser Gly Thr Leu Tyr Lys Tyr Leu Asp Asn Cys Val 575 580 585 agt cca act ggt aag cga ctc tta agg aat tgg atc tgc cat cca ctc 1947 Ser Pro Thr Gly Lys Arg Leu Leu Arg Asn Trp Ile Cys His Pro Leu 590 595 600 aaa gat gta gaa agc atc aat aaa cgg ctt gat gta gtt gaa gaa ttc 1995 Lys Asp Val Glu Ser Ile Asn Lys Arg Leu Asp Val Val Glu Glu Phe 605 610 615 acg gca aac tca gaa agt atg caa atc act ggc cag tat ctc cac aaa 2043 Thr Ala Asn Ser Glu Ser Met Gln Ile Thr Gly Gln Tyr Leu His Lys 620 625 630 ctt cca gac tta gaa aga ctg ctc gga cgc atc aag tct agc gtt cga 2091 Leu Pro Asp Leu Glu Arg Leu Leu Gly Arg Ile Lys Ser Ser Val Arg 635 640 645 650 tca tca gcc tct gtg ttg cct gct ctt ctg ggg aaa aaa gtg ctg aaa 2139 Ser Ser Ala Ser Val Leu Pro Ala Leu Leu Gly Lys Lys Val Leu Lys 655 660 665 caa cga gtt aaa gca ttt ggg caa att gtg aaa ggg ttc aga agt gga 2187 Gln Arg Val Lys Ala Phe Gly Gln Ile Val Lys Gly Phe Arg Ser Gly 670 675 680 att gat ctg ttg ttg gct cta cag aag gaa tca aat atg atg agt ttg 2235 Ile Asp Leu Leu Leu Ala Leu Gln Lys Glu Ser Asn Met Met Ser Leu 685 690 695 ctt tat aaa ctc tgt aaa ctt cct ata tta gta gga aaa agc ggg cta 2283 Leu Tyr Lys Leu Cys Lys Leu Pro Ile Leu Val Gly Lys Ser Gly Leu 700 705 710 gag tta ttt ctt tct caa ttc gaa gca gcc ata gat agc gac ttt cca 2331 Glu Leu Phe Leu Ser Gln Phe Glu Ala Ala Ile Asp Ser Asp Phe Pro 715 720 725 730 aat tat cag aac caa gat gtg aca gat gaa aac gct gaa act ctc aca 2379 Asn Tyr Gln Asn Gln Asp Val Thr Asp Glu Asn Ala Glu Thr Leu Thr 735 740 745 ata ctt atc gaa ctt ttt atc gaa aga gca act caa tgg tct gag gtc 2427 Ile Leu Ile Glu Leu Phe Ile Glu Arg Ala Thr Gln Trp Ser Glu Val 750 755 760 att cac acc ata agc tgc cta gat gtc ctg aga tct ttt gca atc gca 2475 Ile His Thr Ile Ser Cys Leu Asp Val Leu Arg Ser Phe Ala Ile Ala 765 770 775 gca agt ctc tct gct gga agc atg gcc agg cct gtt att ttt ccc gaa 2523 Ala Ser Leu Ser Ala Gly Ser Met Ala Arg Pro Val Ile Phe Pro Glu 780 785 790 tca gaa gct aca gat cag aat cag aaa aca aaa ggg cca ata ctt aaa 2571 Ser Glu Ala Thr Asp Gln Asn Gln Lys Thr Lys Gly Pro Ile Leu Lys 795 800 805 810 atc caa gga cta tgg cat cca ttt gca gtt gca gcc gat ggt caa ttg 2619 Ile Gln Gly Leu Trp His Pro Phe Ala Val Ala Ala Asp Gly Gln Leu 815 820 825 cct gtt ccg aat gat ata ctc ctt ggc gag gct aga aga agc agt ggc 2667 Pro Val Pro Asn Asp Ile Leu Leu Gly Glu Ala Arg Arg Ser Ser Gly 830 835 840 agc att cat cct cgg tca ttg tta ctg acg gga cca aac atg ggc gga 2715 Ser Ile His Pro Arg Ser Leu Leu Leu Thr Gly Pro Asn Met Gly Gly 845 850 855 aaa tca act ctt ctt cgt gca aca tgt ctg gcc gtt atc ttt gcc caa 2763 Lys Ser Thr Leu Leu Arg Ala Thr Cys Leu Ala Val Ile Phe Ala Gln 860 865 870 ctt ggc tgc tac gtg ccg tgt gag tct tgc gaa atc tcc ctc gtg gat 2811 Leu Gly Cys Tyr Val Pro Cys Glu Ser Cys Glu Ile Ser Leu Val Asp 875 880 885 890 act atc ttc aca agg ctt ggc gca tct gat aga atc atg aca gga gag 2859 Thr Ile Phe Thr Arg Leu Gly Ala Ser Asp Arg Ile Met Thr Gly Glu 895 900 905 agt acc ttt ttg gta gaa tgc act gag aca gcg tca gtt ctt cag aat 2907 Ser Thr Phe Leu Val Glu Cys Thr Glu Thr Ala Ser Val Leu Gln Asn 910 915 920 gca act cag gat tca cta gta atc ctt gac gaa ctg ggc aga gga act 2955 Ala Thr Gln Asp Ser Leu Val Ile Leu Asp Glu Leu Gly Arg Gly Thr 925 930 935 agt act ttc gat gga tac gcc att gca tac tcg gtt ttt cgt cac ctg 3003 Ser Thr Phe Asp Gly Tyr Ala Ile Ala Tyr Ser Val Phe Arg His Leu 940 945 950 gta gag aaa gtt caa tgt cgg atg ctc ttt gca aca cat tac cac cct 3051 Val Glu Lys Val Gln Cys Arg Met Leu Phe Ala Thr His Tyr His Pro 955 960 965 970 ctc acc aag gaa ttc gcg tct cac cca cgt gtc acc tcg aaa cac atg 3099 Leu Thr Lys Glu Phe Ala Ser His Pro Arg Val Thr Ser Lys His Met 975 980 985 gct tgc gca ttc aaa tca aga tct gat tat caa cca cgt ggt tgt gat 3147 Ala Cys Ala Phe Lys Ser Arg Ser Asp Tyr Gln Pro Arg Gly Cys Asp 990 995 1000 caa gac cta gtg ttc ttg tac cgt tta acc gag gga gct tgt cct gag 3195 Gln Asp Leu Val Phe Leu Tyr Arg Leu Thr Glu Gly Ala Cys Pro Glu 1005 1010 1015 agc tac gga ctt caa gtg gca ctc atg gct gga ata cca aac caa gtg 3243 Ser Tyr Gly Leu Gln Val Ala Leu Met Ala Gly Ile Pro Asn Gln Val 1020 1025 1030 gtt gaa aca gca tca ggt gct gct caa gcc atg aag aga tca att ggg 3291 Val Glu Thr Ala Ser Gly Ala Ala Gln Ala Met Lys Arg Ser Ile Gly 1035 1040 1045 1050 gga aac ttc aag tca agt gag cta aga tct gag ttc tca agt ctg cat 3339 Glu Asn Phe Lys Ser Ser Glu Leu Arg Ser Glu Phe Ser Ser Leu His 1055 1060 1065 gaa gac tgg ctc aag tca ttg gtg ggt att tct cga gtc gcc cac aac 3387 Glu Asp Trp Leu Lys Ser Leu Val Gly Ile Ser Arg Val Ala His Asn 1070 1075 1080 aat gcc ccc att ggc gaa gat gac tac gac act ttg ttt tgc tta tgg 3435 Asn Ala Pro Ile Gly Glu Asp Asp Tyr Asp Thr Leu Phe Cys Leu Trp 1085 1090 1095 cat gag atc aaa tcc tct tac tgt gtt ccc aaa taaatggcta 3478 His Glu Ile Lys Ser Ser Tyr Cys Val Pro Lys 1100 1105 tgacataaca ctatctgaag ctcgttaagt cttttgcctc tctgatgttt attcctctta 3538 aaaaatgctt atatatcaaa aaattgtttc ctcgattaaa aaaaaaaaaa aaaaaaaaaa 3598 aaaaaaaa 3606 31 1109 PRT Arabidopsis thaliana ecotype Columbia Polypeptide MSH6 31 Met Gln Arg Gln Arg Ser Ile Leu Ser Phe Phe Gln Lys Pro Thr Ala 1 5 10 15 Ala Thr Thr Lys Gly Leu Val Ser Gly Asp Ala Ala Ser Gly Gly Gly 20 25 30 Gly Ser Gly Gly Pro Arg Phe Asn Val Arg Glu Gly Asp Ala Lys Gly 35 40 45 Asp Ala Ser Val Arg Phe Ala Val Ser Lys Ser Val Asp Glu Val Arg 50 55 60 Gly Thr Asp Thr Pro Pro Glu Lys Val Pro Arg Arg Val Leu Pro Ser 65 70 75 80 Gly Phe Lys Pro Ala Glu Ser Ala Gly Asp Ala Ser Ser Leu Phe Ser 85 90 95 Asn Ile Met His Lys Phe Val Lys Val Asp Asp Arg Asp Cys Ser Gly 100 105 110 Glu Arg Ser Arg Glu Asp Val Val Pro Leu Asn Asp Ser Ser Leu Cys 115 120 125 Met Lys Ala Asn Asp Val Ile Pro Gln Phe Arg Ser Asn Asn Gly Lys 130 135 140 Thr Gln Glu Arg Asn His Ala Phe Ser Phe Ser Gly Arg Ala Glu Leu 145 150 155 160 Arg Ser Val Glu Asp Ile Gly Val Asp Gly Asp Val Pro Gly Pro Glu 165 170 175 Thr Pro Gly Met Arg Pro Arg Ala Ser Arg Leu Lys Arg Val Leu Glu 180 185 190 Asp Glu Met Thr Phe Lys Glu Asp Lys Val Pro Val Leu Asp Ser Asn 195 200 205 Lys Arg Leu Lys Met Leu Gln Asp Pro Val Cys Gly Glu Lys Lys Glu 210 215 220 Val Asn Glu Gly Thr Lys Phe Glu Trp Leu Glu Ser Ser Arg Ile Arg 225 230 235 240 Asp Ala Asn Arg Arg Arg Pro Asp Asp Pro Leu Tyr Asp Arg Lys Thr 245 250 255 Leu His Ile Pro Pro Asp Val Phe Lys Lys Met Ser Ala Ser Gln Lys 260 265 270 Gln Tyr Trp Ser Val Lys Ser Glu Tyr Met Asp Ile Val Leu Phe Phe 275 280 285 Lys Val Gly Lys Phe Tyr Glu Leu Tyr Glu Leu Asp Ala Glu Leu Gly 290 295 300 His Lys Glu Leu Asp Trp Lys Met Thr Met Ser Gly Val Gly Lys Cys 305 310 315 320 Arg Gln Val Gly Ile Ser Glu Ser Gly Ile Asp Glu Ala Val Gln Lys 325 330 335 Leu Leu Ala Arg Gly Tyr Lys Val Gly Arg Ile Glu Gln Leu Glu Thr 340 345 350 Ser Asp Gln Ala Lys Ala Arg Gly Ala Asn Thr Ile Ile Pro Arg Lys 355 360 365 Leu Val Gln Val Leu Thr Pro Ser Thr Ala Ser Glu Gly Asn Ile Gly 370 375 380 Pro Asp Ala Val His Leu Leu Ala Ile Lys Glu Ile Lys Met Glu Leu 385 390 395 400 Gln Lys Cys Ser Thr Val Tyr Gly Phe Ala Phe Val Asp Cys Ala Ala 405 410 415 Leu Arg Phe Trp Val Gly Ser Ile Ser Asp Asp Ala Ser Cys Ala Ala 420 425 430 Leu Gly Ala Leu Leu Met Gln Val Ser Pro Lys Glu Val Leu Tyr Asp 435 440 445 Ser Lys Gly Leu Ser Arg Glu Ala Gln Lys Ala Leu Arg Lys Tyr Thr 450 455 460 Leu Thr Gly Ser Thr Ala Val Gln Leu Ala Pro Val Pro Gln Val Met 465 470 475 480 Gly Asp Thr Asp Ala Ala Gly Val Arg Asn Ile Ile Glu Ser Asn Gly 485 490 495 Tyr Phe Lys Gly Ser Ser Glu Ser Trp Asn Cys Ala Val Asp Gly Leu 500 505 510 Asn Glu Cys Asp Val Ala Leu Ser Ala Leu Gly Glu Leu Ile Asn His 515 520 525 Leu Ser Arg Leu Lys Leu Glu Asp Val Leu Lys His Gly Asp Ile Phe 530 535 540 Pro Tyr Gln Val Tyr Arg Gly Cys Leu Arg Ile Asp Gly Gln Thr Met 545 550 555 560 Val Asn Leu Glu Ile Phe Asn Asn Ser Cys Asp Gly Gly Pro Ser Gly 565 570 575 Thr Leu Tyr Lys Tyr Leu Asp Asn Cys Val Ser Pro Thr Gly Lys Arg 580 585 590 Leu Leu Arg Asn Trp Ile Cys His Pro Leu Lys Asp Val Glu Ser Ile 595 600 605 Asn Lys Arg Leu Asp Val Val Glu Glu Phe Thr Ala Asn Ser Glu Ser 610 615 620 Met Gln Ile Thr Gly Gln Tyr Leu His Lys Leu Pro Asp Leu Glu Arg 625 630 635 640 Leu Leu Gly Arg Ile Lys Ser Ser Val Arg Ser Ser Ala Ser Val Leu 645 650 655 Pro Ala Leu Leu Gly Lys Lys Val Leu Lys Gln Arg Val Lys Ala Phe 660 665 670 Gly Gln Ile Val Lys Gly Phe Arg Ser Gly Ile Asp Leu Leu Leu Ala 675 680 685 Leu Gln Lys Glu Ser Asn Met Met Ser Leu Leu Tyr Lys Leu Cys Lys 690 695 700 Leu Pro Ile Leu Val Gly Lys Ser Gly Leu Glu Leu Phe Leu Ser Gln 705 710 715 720 Phe Glu Ala Ala Ile Asp Ser Asp Phe Pro Asn Tyr Gln Asn Gln Asp 725 730 735 Val Thr Asp Glu Asn Ala Glu Thr Leu Thr Ile Leu Ile Glu Leu Phe 740 745 750 Ile Glu Arg Ala Thr Gln Trp Ser Glu Val Ile His Thr Ile Ser Cys 755 760 765 Leu Asp Val Leu Arg Ser Phe Ala Ile Ala Ala Ser Leu Ser Ala Gly 770 775 780 Ser Met Ala Arg Pro Val Ile Phe Pro Glu Ser Glu Ala Thr Asp Gln 785 790 795 800 Asn Gln Lys Thr Lys Gly Pro Ile Leu Lys Ile Gln Gly Leu Trp His 805 810 815 Pro Phe Ala Val Ala Ala Asp Gly Gln 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 Gln Leu Gly Cys Tyr Val Pro 865 870 875 880 Cys Glu Ser Cys Glu Ile Ser Leu Val Asp Thr Ile Phe Thr Arg Leu 885 890 895 Gly Ala Ser Asp Arg Ile Met Thr Gly Glu Ser Thr Phe Leu Val Glu 900 905 910 Cys Thr Glu Thr Ala Ser Val Leu Gln Asn Ala Thr Gln 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 Gln 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 Arg Ser Asp Tyr Gln Pro Arg Gly Cys Asp Gln Asp Leu Val Phe Leu 995 1000 1005 Tyr Arg Leu Thr Glu Gly Ala Cys Pro Glu Ser Tyr Gly Leu Gln Val 1010 1015 1020 Ala Leu Met Ala Gly Ile Pro Asn Gln Val Val Glu Thr Ala Ser Gly 1025 1030 1035 1040 Ala Ala Gln Ala Met Lys Arg Ser Ile Gly Glu Asn Phe Lys Ser Ser 1045 1050 1055 Glu Leu Arg Ser Glu Phe Ser Ser Leu His Glu Asp Trp Leu Lys Ser 1060 1065 1070 Leu Val Gly Ile Ser Arg Val Ala His Asn Asn Ala Pro Ile Gly Glu 1075 1080 1085 Asp Asp Tyr Asp Thr Leu Phe Cys Leu Trp His Glu Ile Lys Ser Ser 1090 1095 1100 Tyr Cys Val Pro Lys 1105 32 24 DNA Artificial sequence Forward primer for PCR amplification of ATHGENEA microsatellite 32 accatgcata gcttaaactt cttg 24 33 22 DNA Artificial sequence Reverse primer for PCR amplification of ATHGENEA microsatellite 33 acataaccac aaataggggt gc 22 34 18 DNA Artificial sequence Forward primer DMCIN-A for PCR on genomic DNA of Arabidopsis thaliana ssp. Landsberg erecta “Ler” 34 gaagcgatat tgttcgtg 18 35 18 DNA Artificial sequence Reverse primer DMCIN-B for PCR on genomic DNA of Arabidopsis thaliana ssp. Landsberg erecta “Ler” 35 agattgcgag aacattcc 18 36 31 DNA Artificial sequence Forward primer DMCIN-1 for PCR on genomic DNA of Arabidopsis thaliana ssp. Landsberg erecta “Ler” 36 acgcgtcgac tcagctatga gattactcgt g 31 37 29 DNA Artificial sequence Reverse primer DMCIN-2 for PCR on genomic DNA of Arabidopsis thaliana ssp. Landsberg erecta “Ler” 37 gctctagatt tctcgctcta agactctct 29 38 32 DNA Artificial sequence Forward primer DMCIN-3 for PCR on genomic DNA of Arabidopsis thaliana ssp. Landsberg erecta “Ler” 38 gctctagagc ttctcttaag taagtgattg at 32 39 48 DNA Artificial sequence Reverse primer DMCIN-4 for PCR on genomic DNA of Arabidopsis thaliana ssp. Landsberg erecta “Ler” 39 tcccccgggc tcgagagatc tccatggttt cttcagctct atgaatcc 48 40 26 DNA Artificial sequence Forward primer DMC1a for PCR on genomic DNA of Arabidopsis thaliana ssp. Landsberg erecta “Ler” 40 acgcgtcgac gaattcgcaa gtgggg 26 41 38 DNA Artificial sequence Reverse primer DMC1b for PCR on genomic DNA of Arabidopsis thaliana ssp. Landsberg erecta “Ler” 41 tccatggaga tctcccgggt accgatttgc ttcgaggg 38 42 20 DNA Artificial sequence Forward primer for PCR amplification of ATEAT1 SSLP marker in Arabidopsis thaliana subspecies 42 gccactgcgt gaatgatatg 20 43 22 DNA Artificial sequence Reverse primer for PCR amplification of ATEAT1 SSLP marker in Arabidopsis thaliana subspecies 43 cgaacagcca acattaattc cc 22 44 18 DNA Artificial sequence Forward primer for PCR amplification of NGA63 SSLP marker in Arabidopsis thaliana subspecies 44 aaccaaggca cagaagcg 18 45 18 DNA Artificial sequence Reverse primer for PCR amplification of NGA63 SSLP marker in Arabidopsis thaliana subspecies 45 acccaagtga tcgccacc 18 46 21 DNA Artificial sequence Forward primer for PCR amplification of NGA248 SSLP marker in Arabidopsis thaliana subspecies 46 taccgaacca aaacacaaag g 21 47 22 DNA Artificial sequence Reverse primer for PCR amplification of NGA248 SSLP marker in Arabidopsis thaliana subspecies 47 tctgtatctc ggtgaattct cc 22 48 22 DNA Artificial sequence Forward primer for PCR amplification of NGA128 SSLP marker in Arabidopsis thaliana subspecies 48 ggtctgttga tgtcgtaagt cg 22 49 22 DNA Artificial sequence Reverse primer for PCR amplification of NGA128 SSLP marker in Arabidopsis thaliana subspecies 49 atcttgaaac ctttagggag gg 22 50 22 DNA Artificial sequence Forward primer for PCR amplification of NGA280 SSLP marker in Arabidopsis thaliana subspecies 50 ctgatctcac ggacaatagt gc 22 51 20 DNA Artificial sequence Reverse primer for PCR amplification of NGA280 SSLP marker in Arabidopsis thaliana subspecies 51 ggctccataa aaagtgcacc 20 52 21 DNA Artificial sequence Forward primer for PCR amplification of NGA111 SSLP marker in Arabidopsis thaliana subspecies 52 ctccagttgg aagctaaagg g 21 53 21 DNA Artificial sequence Reverse primer for PCR amplification of NGA111 SSLP marker in Arabidopsis thaliana subspecies 53 tgttttttag gacaaatggc g 21 54 20 DNA Artificial sequence Forward primer for PCR amplification of NGA168 SSLP marker in Arabidopsis thaliana subspecies 54 ccttcacatc caaaacccac 20 55 20 DNA Artificial sequence Reverse primer for PCR amplification of NGA168 SSLP marker in Arabidopsis thaliana subspecies 55 gcacataccc acaaccagaa 20 56 20 DNA Artificial sequence Forward primer for PCR amplification of NGA1126 SSLP marker in Arabidopsis thaliana subspecies 56 cgctacgctt ttcggtaaag 20 57 20 DNA Artificial sequence Reverse primer for PCR amplification of NGA1126 SSLP marker in Arabidopsis thaliana subspecies 57 gcacagtcca agtcacaacc 20 58 20 DNA Artificial sequence Forward primer for PCR amplification of NGA361 SSLP marker in Arabidopsis thaliana subspecies 58 aaagagatga gaatttggac 20 59 23 DNA Artificial sequence Reverse primer for PCR amplification of NGA361 SSLP marker in Arabidopsis thaliana subspecies 59 acatatcaat atattaaagt agc 23 60 18 DNA Artificial sequence Forward primer for PCR amplification of NGA168 SSLP marker in Arabidopsis thaliana subspecies 60 tcgtctactg cactgccg 18 61 22 DNA Artificial sequence Reverse primer for PCR amplification of NGA168 SSLP marker in Arabidopsis thaliana subspecies 61 gaggacatgt ataggagcct cg 22 62 20 DNA Artificial sequence Forward primer for PCR amplification of AthBIO2 SSLP marker in Arabidopsis thaliana subspecies 62 tgacctcctc ttccatggag 20 63 22 DNA Artificial sequence Reverse primer for PCR amplification of AthBIO2 SSLP marker in Arabidopsis thaliana subspecies 63 ttaacagaaa cccaaagctt tc 22 64 21 DNA Artificial sequence Forward primer for PCR amplification of AthUBIQUE SSLP marker in Arabidopsis thaliana subspecies 64 aggcaaatgt ccatttcatt g 21 65 20 DNA Artificial sequence Reverse primer for PCR amplification of AthUBIQUE SSLP marker in Arabidopsis thaliana subspecies 65 acgacatggc agatttctcc 20 66 21 DNA Artificial sequence Forward primer for PCR amplification of NGA172 SSLP marker in Arabidopsis thaliana subspecies 66 agctgcttcc ttatagcgtc c 21 67 19 DNA Artificial sequence Reverse primer for PCR amplification of NGA172 SSLP marker in Arabidopsis thaliana subspecies 67 catccgaatg ccattgttc 19 68 21 DNA Artificial sequence Forward primer for PCR amplification of NGA126 SSLP marker in Arabidopsis thaliana subspecies 68 gaaaaaacgc tactttcgtg g 21 69 22 DNA Artificial sequence Reverse primer for PCR amplification of NGA126 SSLP marker in Arabidopsis thaliana subspecies 69 caagagcaat atcaagagca gc 22 70 20 DNA Artificial sequence Forward primer for PCR amplification of NGA162 SSLP marker in Arabidopsis thaliana subspecies 70 catgcaattt gcatctgagg 20 71 22 DNA Artificial sequence Reverse primer for PCR amplification of NGA162 SSLP marker in Arabidopsis thaliana subspecies 71 ctctgtcact cttttcctct gg 22 72 21 DNA Artificial sequence Forward primer for PCR amplification of NGA6 SSLP marker in Arabidopsis thaliana subspecies 72 tggatttctt cctctcttca c 21 73 21 DNA Artificial sequence Reverse primer for PCR amplification of NGA6 SSLP marker in Arabidopsis thaliana subspecies 73 atggagaagc ttacactgat c 21 74 20 DNA Artificial sequence Forward primer for PCR amplification of NGA12 SSLP marker in Arabidopsis thaliana subspecies 74 aatgttgtcc tcccctcctc 20 75 22 DNA Artificial sequence Reverse primer for PCR amplification of NGA12 SSLP marker in Arabidopsis thaliana subspecies 75 tgatgctctc tgaaacaaga gc 22 76 21 DNA Artificial sequence Forward primer for PCR amplification of NGA8 SSLP marker in Arabidopsis thaliana subspecies 76 gagggcaaat ctttatttcg g 21 77 22 DNA Artificial sequence Reverse primer for PCR amplification of NGA8 SSLP marker in Arabidopsis thaliana subspecies 77 tggctttcgt ttataaacat cc 22 78 21 DNA Artificial sequence Forward primer for PCR amplification of NGA1107 SSLP marker in Arabidopsis thaliana subspecies 78 gcgaaaaaac aaaaaaatcc a 21 79 21 DNA Artificial sequence Reverse primer for PCR amplification of NGA1107 SSLP marker in Arabidopsis thaliana subspecies 79 cgacgaatcg acagaattag g 21 80 21 DNA Artificial sequence Forward primer for PCR amplification of NGA225 SSLP marker in Arabidopsis thaliana subspecies 80 gaaatccaaa tcccagagag g 21 81 22 DNA Artificial sequence Reverse primer for PCR amplification of NGA225 SSLP marker in Arabidopsis thaliana subspecies 81 tctccccact agttttgtgt cc 22 82 19 DNA Artificial sequence Forward primer for PCR amplification of NGA249 SSLP marker in Arabidopsis thaliana subspecies 82 taccgtcaat ttcatcgcc 19 83 22 DNA Artificial sequence Reverse primer for PCR amplification of NGA249 SSLP marker in Arabidopsis thaliana subspecies 83 ggatccctaa ctgtaaaatc cc 22 84 22 DNA Artificial sequence Forward primer for PCR amplification of CA72 SSLP marker in Arabidopsis thaliana subspecies 84 aatcccagta accaaacaca ca 22 85 20 DNA Artificial sequence Reverse primer for PCR amplification of CA72 SSLP marker in Arabidopsis thaliana subspecies 85 cccagtctaa ccacgaccac 20 86 20 DNA Artificial sequence Forward primer for PCR amplification of NGA151 SSLP marker in Arabidopsis thaliana subspecies 86 gttttgggaa gttttgctgg 20 87 24 DNA Artificial sequence Reverse primer for PCR amplification of NGA151 SSLP marker in Arabidopsis thaliana subspecies 87 cagtctaaaa gcgagagtat gatg 24 88 22 DNA Artificial sequence Forward primer for PCR amplification of NGA106 SSLP marker in Arabidopsis thaliana subspecies 88 gttatggagt ttctagggca cg 22 89 20 DNA Artificial sequence Reverse primer for PCR amplification of NGA106 SSLP marker in Arabidopsis thaliana subspecies 89 tgccccattt tgttcttctc 20 90 20 DNA Artificial sequence Forward primer for PCR amplification of NGA139 SSLP marker in Arabidopsis thaliana subspecies 90 agagctacca gatccgatgg 20 91 21 DNA Artificial sequence Reverse primer for PCR amplification of NGA139 SSLP marker in Arabidopsis thaliana subspecies 91 ggtttcgttt cactatccag g 21 92 22 DNA Artificial sequence Forward primer for PCR amplification of NGA76 SSLP marker in Arabidopsis thaliana subspecies 92 ggagaaaatg tcactctcca cc 22 93 20 DNA Artificial sequence Reverse primer for PCR amplification of NGA76 SSLP marker in Arabidopsis thaliana subspecies 93 aggcatggga gacatttacg 20 94 20 DNA Artificial sequence Forward primer for PCR amplification of ATHSO191 SSLP marker in Arabidopsis thaliana subspecies 94 ctccaccaat catgcaaatg 20 95 21 DNA Artificial sequence Reverse primer for PCR amplification of ATHSO191 SSLP marker in Arabidopsis thaliana subspecies 95 tgatgttgat ggagatggtc a 21 96 22 DNA Artificial sequence Forward primer for PCR amplification of NGA129 SSLP marker in Arabidopsis thaliana subspecies 96 tcaggaggaa ctaaagtgag gg 22 97 22 DNA Artificial sequence Reverse primer for PCR amplification of NGA129 SSLP marker in Arabidopsis thaliana subspecies 97 cacactgaag atggtcttga gg 22 98 8062 DNA Arabidopsis thaliana ecotype Columbia Genomic DNA sequence of AtMSH6 98 ttttttggtt gctaacaata aaggtatacg gttttatgtc atcaatataa ctatatataa 60 aagaaatgaa agatatatat tgttttttca tttatcaaac aaaacaacaa gacttttttt 120 ttacttttta cattggtcaa caaaatacaa gataaacgac atcgtttaat catttcccaa 180 ttttacccct aagtttaaca cctagaacct tctccatctt cgcaagcaca gcctgattag 240 gaacagcttt accattctca tattcctgaa ctacctgagt cctctcattg atctgtttcg 300 ccaaatccgc ttgtgacatc ttcttctcca atctcgcttt ctgtatcatc aacctcacct 360 ctgctttcac acgatccatc gccgcaggct ctgtttcttc ttccagcttc ttcgtgttaa 420 tcaccggaac cgccgtagat ttcccctttt tgttcgaacc ggcatcgaat ttcttaaccg 480 tttgaaccgc gacaccgttt ctcagagctg cgttaaccgc tttcggatcg cgtaggtctt 540 ggctcttttg ttttgatttg tggagaacta ctggttccca gtcttgtgtt actgctcctg 600 ggtatctgct cggcatcgtc gatgaattga gagaaaggaa caacgcgaaa attttattaa 660 tctgagtttt gaaattgaga aacgatgaag atgaagaatg ttgttgagag gattgtgata 720 tttatatata cgaagattgg tttctggaga attcgatcat ctttttctcc attttcgtct 780 ctggaacgtt cttagagatg attgacgacg tgtcattatc tgatttgcag ttaaccaatg 840 ctttttgggt tggattcgtg gtacaccata ttatccgatt tggctcaatg gttttatata 900 aatttggttt tcggttcggt tatgagttat cattaaaatt aagctaacca aaaattttcg 960 taaaatttat ttcggtttca attcggatcc cttacttcca gaaccgaatt attcgaaacc 1020 ggggttagcc gaaccgaata ccaatgcctg attgactcgt tggctagaaa gatccaacgg 1080 tatacaataa tagaacataa atcggacggt catcaaagcc tcaaagagtg aacagtcaac 1140 aaaaaaagtt gagccctgag gagtatcgtt tccgccattt ctacgacgca aggcgaaaat 1200 ttttggcgcc aatctttccc ccctttcgaa ttctctcagc tcaaaacatc gtttctctct 1260 cactctctct cacaattcca aaaaatgcag cgccagagat cgattttgtc tttcttccaa 1320 aaacccacgg cggcgactac gaagggtttg gtttccggcg atgctgctag cggcgggggc 1380 ggcagcggag accacgattt aatgtgaagg aaggggatgc taaaggcgac gcttctgtac 1440 gttttgctgt ttcgaaatct gtcgatgagg ttagaggaac ggatactcca ccggagaagg 1500 ttccgcgtcg tgtcctgccg tctggattta agccggctga atccgccggt gatgcttcgt 1560 ccctgttctc caatattatg cataagtttg taaaagtcga tgatcgagat tgttctggag 1620 agaggtacta atcttcgatt ctcttaattt tgttatcttt agctggaaga agaagattcg 1680 tgtaatttgt tgtattcgtt ggagagattc tgattactgc attggatcgt tgtttacaaa 1740 ttttcaggag ccgagaagat gttgttccgc tgaatgattc atctctatgt atgaaggcta 1800 atgatgttat tcctcaattt cgttccaata atggtaaaac tcaagaaaga aaccatgctt 1860 ttagtttcag tgggagagct gaacttagat cagtagaaga tataggagta gatggcgatg 1920 ttcctggtcc agaaacacca gggatgcgtc cacgtgcttc tcgcttgaag cgagttctgg 1980 aggatgaaat gacttttaag gaggataagg ttcctgtatt ggactctaac aaaaggctga 2040 aaatgctcca ggatccggtt tgtggagaga agaaagaagt aaacgaagga accaaatttg 2100 aatggcttga gtcttctcga atcagggatg ccaatagaag acgtcctgat gatccccttt 2160 acgatagaaa gaccttacac ataccacctg atgttttcaa gaaaatgtct gcatcacaaa 2220 agcaatattg gagtgttaag agtgaatata tggacattgt gcttttcttt aaagtggtta 2280 gtaactatta atctagtgtt caatccattt cctcaatgtg atttgttcac ttacatctgt 2340 ttacgttatg ctcttctcag gggaaatttt atgagctgta tgagctagat gcggaattag 2400 gtcacaagga gcttgactgg aagatgacca tgagtggtgt gggaaaatgc agacaggtaa 2460 attagttgaa acaactggcc tgcttgaatt attgtgtcta taaattttga caccaccttt 2520 tgtttcaggt tggtatctct gaaagtggga tagatgaggc agtgcaaaag ctattagctc 2580 gtgggtaagg gaaccatcat actttatgga attcgtttac tgctacttcg gctaggattt 2640 aagaaatgga aatcacttca agcatcatta gttaggatcc tgagaactca ggatgttttc 2700 ttattcgtta tataataagt cttttcatca aggagtaaca aacaaaactt gcacaatatt 2760 tgtgtgctca ctggcaaggc atatataccc agctaacctt tgctagttca ctgtagtaac 2820 agttacggat aatatatgtt tacttgtatg tggtaccctc attttgtctc tcatggaggc 2880 tttcaagcct tgtgttgaaa ctggatagtt acatatgctt ccaacagaaa ctagcatgca 2940 gattcatatg ctttcctatt ctactaatta tgtattgaca cactcgttgt ttcttttgaa 3000 agatataaag ttggacgaat cgagcagcta gaaacatctg accaagcaaa agccagaggt 3060 gctaatactg taagttttct tggataggtc aaggagagtg ttgcagactg tttttgatca 3120 tttctttttc tgtacattac tttcatgctg taattaactc aatggctatt ctggtctgat 3180 tatcagataa ttccaaggaa gctagttcag gtattaactc catcaacagc aagcgaggga 3240 aacatcgggc ctgatgccgt ccatcttctt gctataaaag aggtttgtta tttacttatt 3300 tatcttatca tgttcagttc atccaagtcc tgaaaaatta cactcttctt taccaatctt 3360 ccatcaagct gtgtaaagga tttggaatta gaaaatcatt atttgatgct ttgttttata 3420 tgcaagaggt tcccttgaaa agatctgttt aagattcttt gcacttgaaa aattcaatct 3480 ttttaagtga atcccctact ttcttacaat gatcatagtc tgcaattgca tgtcaagtaa 3540 tatcattcct tgttactgca tccccctctt tcttaatgac cattgtctat gttgtgtttg 3600 tctcgtgtgc tggagaaaat gatagctgat ccaagctgta cattatcatg attaagtagc 3660 tgctcaggaa ttgcctttgg ttacattgcc taatggtttg atgtcaattt ttcttctgaa 3720 tctttatttt agatcaaaat ggagctacaa aagtgttcaa ctgtgtatgg atttgctttt 3780 gttgactgtg ctgccttgag gttttgggtt gggtccatca gcgatgatgc atcatgtgct 3840 gctcttggag cgttattgat gcaggtaagc aagtgtattc tgtatcttat gtgtaccatg 3900 tgacttcctg tgcatatatt tgggttgcag gaactaattc tgaatcacca tttggtatgt 3960 tttttccagg tttctccaaa ggaagtgtta tatgacagta aaggtaaact gcttgtatcg 4020 ccagttgttt tgttaaacag aatttaaggt aaatgacact ggttaattta aagtgcatac 4080 atgttgaaat attgcagggc tatcaagaga agcacaaaag gctctaagga aatatacgtt 4140 gacaggtacc atttcagtag gcaagctaac tgacaattta accgctcacc gaatgatagg 4200 tctcttaaac attgctaatg tagatgatgt ttatgtttca atctaatagg gtctacggcg 4260 gtacagttgg ctccagtacc acaagtaatg ggggatacag atgctgctgg agttagaaat 4320 ataatagaat ctaacggata ctttaaaggt tcttctgaat catggaactg tgctgttgat 4380 ggtctaaatg aatgtgatgt tgcccttagt gctcttggag agctaattaa tcatctgtct 4440 aggctaaagg tgtgttggct tgtttagttt ttgcttttca caaattaagc aaaggaactt 4500 ttcataactt acagtttcta tctacttgca gctagaagat gtacttaagc atggggatat 4560 ttttccatac caagtttaca ggggttgtct cagaattgat ggccagacga tggtaaatct 4620 tgagatattt aacaatagct gtgatggtgg tccttcaggc aagtgcatat ttcttttttg 4680 ataacttcaa ctagagggca gacatagaag gaaaaattct aatacttcgt acggatctcc 4740 agtaagtaat agccgatttt tgtttaccta tgtagggacc ttgtacaaat atcttgataa 4800 ctgtgttagt ccaactggta agcgactctt aaggaattgg atctgccatc cactcaaaga 4860 tgtagaaagc atcaataaac ggcttgatgt agttgaagaa ttcacggcaa actcagaaag 4920 tatgcaaatc actggccagt atctccacaa acttccagac ttagaaagac tgctcggacg 4980 catcaagtct agcgttcgat catcagcctc tgtgttgcct gctcttctgg ggaaaaaagt 5040 gctgaaacaa cgagtaagta tcaatcacaa gttttctgag taatgccttc catgagtagt 5100 ataggactaa aacattacgg gtctagctaa agactgttct ccttcttttg caatgtctgg 5160 ttattcatta catttctctt aacttattgc attgcaggtt aaagcatttg ggcaaattgt 5220 gaaagggttc agaagtggaa ttgatctgtt gttggctcta cagaaggaat caaatatgat 5280 gagtttgctt tataaactct gtaaacttcc tatattagta ggaaaaagcg ggctagagtt 5340 atttctttct caattcgaag cagccataga tagcgacttt ccaaattatc aggtgcccat 5400 ctatctttca tactttacaa caaaatgtct gtcactactc aaagcaatgc atatggctta 5460 gatctcaact cacaccccga ggatcctaaa gggatttgct ttttattcct aatgtttttg 5520 gatggtttga tttatttcta acttgaactt attaatcttg taccagaacc aagatgtgac 5580 agatgaaaac gctgaaactc tcacaatact tatcgaactt tttatcgaaa gagcaactca 5640 atggtctgag gtcattcaca ccataagctg cctagatgtc ctgagatctt ttgcaatcgc 5700 agcaagtctc tctgctggaa gcatggccag gcctgttatt tttcccgaat cagaagctac 5760 agatcagaat cagaaaacaa aagggccaat acttaaaatc caaggactat ggcatccatt 5820 tgcagttgca gccgatggtc aattgcctgt tccgaatgat atactccttg gcgaggctag 5880 aagaagcagt ggcagcattc atcctcggtc attgttactg acgggaccaa acatgggcgg 5940 aaaatcaact cttcttcgtg caacatgtct ggccgttatc tttgcccaag tttgtatact 6000 cgttagataa ttactctatt ctttgcaatc agttcttcaa catgaataat aaattctgtt 6060 ttctgtctgc agcttggctg ctacgtgccg tgtgagtctt gcgaaatctc cctcgtggat 6120 actatcttca caaggcttgg cgcatctgat agaatcatga caggagagag taagttttgt 6180 tctcaaaata ccaattcctc gaactattta ctcagatttt gtctgattgg acaaggtggt 6240 tttgcttttt tttaggtacc tttttggtag aatgcactga gacagcgtca gttcttcaga 6300 atgcaactca ggattcacta gtaatccttg acgaactggg cagaggaact agtactttcg 6360 atggatacgc cattgcatac tcggtaacct gctcttctcc ttcaacttat acttgttgat 6420 caacaaaaac atgcaattca ttttgctgaa acttattgat ttatatcagg tttttcgtca 6480 cctggtagag aaagttcaat gtcggatgct ctttgcaaca cattaccacc ctctcaccaa 6540 ggaattcgcg tctcacccac gtgtcacctc gaaacacatg gcttgcgcat tcaaatcaag 6600 atctgattat caaccacgtg gttgtgatca agacctagtg ttcttgtacc gtttaaccga 6660 gggagcttgt cctgagagct acggacttca agtggcactc atggctggaa taccaaacca 6720 agtggttgaa acagcatcag gtgctgctca agccatgaag agatcaattg gggaaaactt 6780 caagtcaagt gagctaagat ctgagttctc aagtctgcat gaagactggc tcaagtcatt 6840 ggtgggtatt tctcgagtcg cccacaacaa tgcccccatt ggcgaagatg actacgacac 6900 tttgttttgc ttatggcatg agatcaaatc ctcttactgt gttcccaaat aaatggctat 6960 gacataacac tatctgaagc tcgttaagtc ttttgcttct ctgatgttta ttcctcttaa 7020 aaaatgctta tatatcaaaa aattgtttcc tcgattataa caagattata tatgtatctg 7080 tcggtttagc tatggtatat aatatatgta tgttcatgag attggtcaag agaaatactc 7140 acaaacagta tattaagaag gaaatatgtt tatgcattaa tttaagtttc aagataaact 7200 gcaaataacc tcgactaaag ttgcaaagac caaacacaaa ttacaaaact tataagactt 7260 aagttctgaa ttccctaaaa ccaaaaaaaa aaacagaaca tattttgttg catctacaaa 7320 caacacaaac ctacatagtt tataacttac tcatcactga gattaacatc agaatcattc 7380 tccatttctt catcttcact ctcatcatca tcaccaccac catgatgatt ctcctcctct 7440 tcacgtaacc tagcaatctc actctgagct ctatcaacaa tctgcttctt ctgcaactcc 7500 aaatctctct gaaaatcagc tctcatcttc tccaactcct tcatttgctc tttcttactc 7560 ttctccatct tctcataaac cttcccaaac ctctcaacag aatccgccaa catcttatac 7620 gaagcagcgt cattaacctt cttcctctcg tactcaacct catcatcctc atcctcctcc 7680 tcttcagaat caccaggact atccatcatc tcatcaaacc cattagactt atctaaataa 7740 accttagtgt tcataaacac aaactcacct gaatcaacac cacaagctaa acctaaatcc 7800 gacttgggcg aaacacaaag caacatatcc aacttattga aaaacgacca tttacttgaa 7860 cctaaacctg atttctcaac cttaatcttc tcttttctat acttcctctt caagtcatca 7920 atcattctcc tacattgcgt ctcagatttc tccatcctta gctcctcact cactttctca 7980 gctacttcat tccaatcctc gttcctcaaa ctccttctac ccaattgcaa aaacctatct 8040 ccccaaactt caagcaacac aa 8062 99 5 PRT Arabidopsis thaliana PEPTIDE (1)...(5) Positions 816-820 of AtMSH3 and 852-856 of AtMSH6 99 Thr Gly Pro Asn Met 1 5 100 5 PRT Arabidopsis thaliana PEPTIDE (1)...(5) Positions 964-968 of AtMSH6 100 Phe Ala Thr His Tyr 1 5 101 5 PRT Arabidopsis thaliana PEPTIDE (1)...(5) Positions 928-932 of AtMSH3 101 Phe Val Thr His Tyr 1 5 102 1047 PRT Saccharomyces cerevisiae 102 Met Val Ile Gly Asn Glu Pro Lys Leu Val Leu Leu Arg Ala Lys Ser 1 5 10 15 Ser Ala Asn Arg Phe Ile Leu Leu Asn Leu Leu Thr Ile Met Ala Gly 20 25 30 Gln Pro Thr Ile Ser Arg Phe Phe Lys Lys Ala Val Lys Ser Glu Leu 35 40 45 Thr His Lys Gln Glu Gln Glu Val Ala Val Gly Asn Gly Ala Gly Ser 50 55 60 Glu Ser Ile Cys Leu Asp Thr Asp Glu Glu Asp Asn Leu Ser Ser Val 65 70 75 80 Ala Ser Thr Thr Val Thr Asn Asp Ser Phe Pro Leu Lys Gly Ser Val 85 90 95 Ser Ser Lys Asn Ser Lys Asn Ser Glu Lys Thr Ser Gly Thr Ser Thr 100 105 110 Thr Phe Asn Asp Ile Asp Phe Ala Lys Lys Leu Asp Arg Ile Met Lys 115 120 125 Arg Arg Ser Asp Glu Asn Val Glu Ala Glu Asp Asp Glu Glu Glu Gly 130 135 140 Glu Glu Asp Phe Val Lys Lys Lys Ala Arg Lys Ser Pro Thr Ala Lys 145 150 155 160 Leu Thr Pro Leu Asp Lys Gln Val Lys Asp Leu Lys Met His His Arg 165 170 175 Asp Lys Val Leu Val Ile Arg Val Gly Tyr Lys Tyr Lys Cys Phe Ala 180 185 190 Glu Asp Ala Val Thr Val Ser Arg Ile Leu His Ile Lys Leu Val Pro 195 200 205 Gly Lys Leu Thr Ile Asp Glu Ser Asn Pro Gln Asp Cys Asn His Arg 210 215 220 Gln Phe Ala Tyr Cys Ser Phe Pro Asp Val Arg Leu Asn Val His Leu 225 230 235 240 Glu Arg Leu Val His His Asn Leu Lys Val Ala Val Val Glu Gln Ala 245 250 255 Glu Thr Ser Ala Ile Lys Lys His Asp Pro Gly Ala Ser Lys Ser Ser 260 265 270 Val Phe Glu Arg Lys Ile Ser Asn Val Phe Thr Lys Ala Thr Phe Gly 275 280 285 Val Asn Ser Thr Phe Val Leu Arg Gly Lys Arg Ile Leu Gly Asp Thr 290 295 300 Asn Ser Ile Trp Ala Leu Ser Arg Asp Val His Gln Gly Lys Val Ala 305 310 315 320 Lys Tyr Ser Leu Ile Ser Val Asn Leu Asn Asn Gly Glu Val Val Tyr 325 330 335 Asp Glu Phe Glu Glu Pro Asn Leu Ala Asp Glu Lys Leu Gln Ile Arg 340 345 350 Ile Lys Tyr Leu Gln Pro Ile Glu Val Leu Val Asn Thr Asp Asp Leu 355 360 365 Pro Leu His Val Ala Lys Phe Phe Lys Asp Ile Ser Cys Pro Leu Ile 370 375 380 His Lys Gln Glu Tyr Asp Leu Glu Asp His Val Val Gln Ala Ile Lys 385 390 395 400 Val Met Asn Glu Lys Ile Gln Leu Ser Pro Ser Leu Ile Arg Leu Val 405 410 415 Ser Lys Leu Tyr Ser His Met Val Glu Tyr Asn Asn Glu Gln Val Met 420 425 430 Leu Ile Pro Ser Ile Tyr Ser Pro Phe Ala Ser Lys Ile His Met Leu 435 440 445 Leu Asp Pro Asn Ser Leu Gln Ser Leu Asp Ile Phe Thr His Asp Gly 450 455 460 Gly Lys Gly Ser Leu Phe Trp Leu Leu Asp His Thr Arg Thr Ser Phe 465 470 475 480 Gly Leu Arg Met Leu Arg Glu Trp Ile Leu Lys Pro Leu Val Asp Val 485 490 495 His Gln Ile Glu Glu Arg Leu Asp Ala Ile Glu Cys Ile Thr Ser Glu 500 505 510 Ile Asn Asn Ser Ile Phe Phe Glu Ser Leu Asn Gln Met Leu Asn His 515 520 525 Thr Pro Asp Leu Leu Arg Thr Leu Asn Arg Ile Met Tyr Gly Thr Thr 530 535 540 Ser Arg Lys Glu Val Tyr Phe Tyr Leu Lys Gln Ile Thr Ser Phe Val 545 550 555 560 Asp His Phe Lys Met His Gln Ser Tyr Leu Ser Glu His Phe Lys Ser 565 570 575 Ser Asp Gly Arg Ile Gly Lys Gln Ser Pro Leu Leu Phe Arg Leu Phe 580 585 590 Ser Glu Leu Asn Glu Leu Leu Ser Thr Thr Gln Leu Pro His Phe Leu 595 600 605 Thr Met Ile Asn Val Ser Ala Val Met Glu Lys Asn Ser Asp Lys Gln 610 615 620 Val Met Asp Phe Phe Asn Leu Asn Asn Tyr Asp Cys Ser Glu Gly Ile 625 630 635 640 Ile Lys Ile Gln Arg Glu Ser Glu Ser Val Arg Ser Gln Leu Lys Glu 645 650 655 Glu Leu Ala Glu Ile Arg Lys Tyr Leu Lys Arg Pro Tyr Leu Asn Phe 660 665 670 Arg Asp Glu Val Asp Tyr Leu Ile Glu Val Lys Asn Ser Gln Ile Lys 675 680 685 Asp Leu Pro Asp Asp Trp Ile Lys Val Asn Asn Thr Lys Met Val Ser 690 695 700 Arg Phe Thr Thr Pro Arg Thr Gln Lys Leu Thr Gln Lys Leu Glu Tyr 705 710 715 720 Tyr Lys Asp Leu Leu Ile Arg Glu Ser Glu Leu Gln Tyr Lys Glu Phe 725 730 735 Leu Asn Lys Ile Thr Ala Glu Tyr Thr Glu Leu Arg Lys Ile Thr Leu 740 745 750 Asn Leu Ala Gln Tyr Asp Cys Ile Leu Ser Leu Ala Ala Thr Ser Cys 755 760 765 Asn Val Asn Tyr Val Arg Pro Thr Phe Val Asn Gly Gln Gln Ala Ile 770 775 780 Ile Ala Lys Asn Ala Arg Asn Pro Ile Ile Glu Ser Leu Asp Val His 785 790 795 800 Tyr Val Pro Asn Asp Ile Met Met Ser Pro Glu Asn Gly Lys Ile Asn 805 810 815 Ile Ile Thr Gly Pro Asn Met Gly Gly Lys Ser Ser Tyr Ile Arg Gln 820 825 830 Val Ala Leu Leu Thr Ile Met Ala Gln Ile Gly Ser Phe Val Pro Ala 835 840 845 Glu Glu Ile Arg Leu Ser Ile Phe Glu Asn Val Leu Thr Arg Ile Gly 850 855 860 Ala His Asp Asp Ile Ile Asn Gly Asp Ser Thr Phe Lys Val Glu Met 865 870 875 880 Leu Asp Ile Leu His Ile Leu Lys Asn Cys Asn Lys Arg Ser Leu Leu 885 890 895 Leu Leu Asp Glu Val Gly Arg Gly Thr Gly Thr His Asp Gly Ile Ala 900 905 910 Ile Ser Tyr Ala Leu Ile Lys Tyr Phe Ser Glu Leu Ser Asp Cys Pro 915 920 925 Leu Ile Leu Phe Thr Thr His Phe Pro Met Leu Gly Glu Ile Lys Ser 930 935 940 Pro Leu Ile Arg Asn Tyr His Met Asp Tyr Val Glu Glu Gln Lys Thr 945 950 955 960 Gly Glu Asp Trp Met Ser Val Ile Phe Leu Tyr Lys Leu Lys Lys Gly 965 970 975 Leu Thr Tyr Asn Ser Tyr Gly Met Asn Val Ala Lys Leu Ala Arg Leu 980 985 990 Asp Lys Asp Ile Ile Asn Arg Ala Phe Ser Ile Ser Glu Glu Leu Arg 995 1000 1005 Lys Glu Ser Ile Asn Glu Asp Ala Leu Lys Leu Phe Ser Ser Leu Lys 1010 1015 1020 Arg Ile Leu Lys Ser Asp Asn Ile Thr Ala Thr Asp Lys Leu Ala Lys 1025 1030 1035 1040 Leu Leu Ser Leu Asp Ile His 1045 103 1242 PRT Saccharomyces cerevisiae 103 Met Ala Pro Ala Thr Pro Lys Thr Ser Lys Thr Ala His Phe Glu Asn 1 5 10 15 Gly Ser Thr Ser Ser Gln Lys Lys Met Lys Gln Ser Ser Leu Leu Ser 20 25 30 Phe Phe Ser Lys Gln Val Pro Ser Gly Thr Pro Ser Lys Lys Val Gln 35 40 45 Lys Pro Thr Pro Ala Thr Leu Glu Asn Thr Ala Thr Asp Lys Ile Thr 50 55 60 Lys Asn Pro Gln Gly Gly Lys Thr Gly Lys Leu Phe Val Asp Val Asp 65 70 75 80 Glu Asp Asn Asp Leu Thr Ile Ala Glu Glu Thr Val Ser Thr Val Arg 85 90 95 Ser Asp Ile Met His Ser Gln Glu Pro Gln Ser Asp Thr Met Leu Asn 100 105 110 Ser Asn Thr Thr Glu Pro Lys Ser Thr Thr Thr Asp Glu Asp Leu Ser 115 120 125 Ser Ser Gln Ser Arg Arg Asn His Lys Arg Arg Val Asn Tyr Ala Glu 130 135 140 Ser Asp Asp Asp Asp Ser Asp Thr Thr Phe Thr Ala Lys Arg Lys Lys 145 150 155 160 Gly Lys Val Val Asp Ser Glu Ser Asp Glu Asp Glu Tyr Leu Pro Asp 165 170 175 Lys Asn Asp Gly Asp Glu Asp Asp Asp Ile Ala Asp Asp Lys Glu Asp 180 185 190 Ile Lys Gly Glu Leu Ala Glu Asp Ser Gly Asp Asp Asp Asp Leu Ile 195 200 205 Ser Leu Ala Glu Thr Thr Ser Lys Lys Lys Phe Ser Tyr Asn Thr Ser 210 215 220 His Ser Ser Ser Pro Phe Thr Arg Asn Ile Ser Arg Asp Asn Ser Lys 225 230 235 240 Lys Lys Ser Arg Pro Asn Gln Ala Pro Ser Arg Ser Tyr Asn Pro Ser 245 250 255 His Ser Gln Pro Ser Ala Thr Ser Lys Ser Ser Lys Phe Asn Lys Gln 260 265 270 Asn Glu Glu Arg Tyr Gln Trp Leu Val Asp Glu Arg Asp Ala Gln Arg 275 280 285 Arg Pro Lys Ser Asp Pro Glu Tyr Asp Pro Arg Thr Leu Tyr Ile Pro 290 295 300 Ser Ser Ala Trp Asn Lys Phe Thr Pro Phe Glu Lys Gln Tyr Trp Glu 305 310 315 320 Ile Lys Ser Lys Met Trp Asp Cys Ile Val Phe Phe Lys Lys Gly Lys 325 330 335 Phe Phe Glu Leu Tyr Glu Lys Asp Ala Leu Leu Ala Asn Ala Leu Phe 340 345 350 Asp Leu Lys Ile Ala Gly Gly Gly Arg Ala Asn Met Gln Leu Ala Gly 355 360 365 Ile Pro Glu Met Ser Phe Glu Tyr Trp Ala Ala Gln Phe Ile Gln Met 370 375 380 Gly Tyr Lys Val Ala Lys Val Asp Gln Arg Glu Ser Met Leu Ala Lys 385 390 395 400 Glu Met Arg Glu Gly Ser Lys Gly Ile Val Lys Arg Glu Leu Gln Cys 405 410 415 Ile Leu Thr Ser Gly Thr Leu Thr Asp Gly Asp Met Leu His Ser Asp 420 425 430 Leu Ala Thr Phe Cys Leu Ala Ile Arg Glu Glu Pro Gly Asn Phe Tyr 435 440 445 Asn Glu Thr Gln Leu Asp Ser Ser Thr Ile Val Gln Lys Leu Asn Thr 450 455 460 Lys Ile Phe Gly Ala Ala Phe Ile Asp Thr Ala Thr Gly Glu Leu Gln 465 470 475 480 Met Leu Glu Phe Glu Asp Asp Ser Glu Cys Thr Lys Leu Asp Thr Leu 485 490 495 Met Ser Gln Val Arg Pro Met Glu Val Val Met Glu Arg Asn Asn Leu 500 505 510 Ser Thr Leu Ala Asn Lys Ile Val Lys Phe Asn Ser Ala Pro Asn Ala 515 520 525 Ile Phe Asn Glu Val Lys Ala Gly Glu Glu Phe Tyr Asp Cys Asp Lys 530 535 540 Thr Tyr Ala Glu Ile Ile Ser Ser Glu Tyr Phe Ser Thr Glu Glu Asp 545 550 555 560 Trp Pro Glu Val Leu Lys Ser Tyr Tyr Asp Thr Gly Lys Lys Val Gly 565 570 575 Phe Ser Ala Phe Gly Gly Leu Leu Tyr Tyr Leu Lys Trp Leu Lys Leu 580 585 590 Asp Lys Asn Leu Ile Ser Met Lys Asn Ile Lys Glu Tyr Asp Phe Val 595 600 605 Lys Ser Gln His Ser Met Val Leu Asp Gly Ile Thr Leu Gln Asn Leu 610 615 620 Glu Ile Phe Ser Asn Ser Phe Asp Gly Ser Asp Lys Gly Thr Leu Phe 625 630 635 640 Lys Leu Phe Asn Arg Ala Ile Thr Pro Met Gly Lys Arg Met Met Lys 645 650 655 Lys Trp Leu Met His Pro Leu Leu Arg Lys Asn Asp Ile Glu Ser Arg 660 665 670 Leu Asp Ser Val Asp Ser Leu Leu Gln Asp Ile Thr Leu Arg Glu Gln 675 680 685 Leu Glu Ile Thr Phe Ser Lys Leu Pro Asp Leu Glu Arg Met Leu Ala 690 695 700 Arg Ile His Ser Arg Thr Ile Lys Val Lys Asp Phe Glu Lys Val Ile 705 710 715 720 Thr Ala Phe Glu Thr Ile Ile Glu Leu Gln Asp Ser Leu Lys Asn Asn 725 730 735 Asp Leu Lys Gly Asp Val Ser Lys Tyr Ile Ser Ser Phe Pro Glu Gly 740 745 750 Leu Val Glu Ala Val Lys Ser Trp Thr Asn Ala Phe Glu Arg Gln Lys 755 760 765 Ala Ile Asn Glu Asn Ile Ile Val Pro Gln Arg Gly Phe Asp Ile Glu 770 775 780 Phe Asp Lys Ser Met Asp Arg Ile Gln Glu Leu Glu Asp Glu Leu Met 785 790 795 800 Glu Ile Leu Met Thr Tyr Arg Lys Gln Phe Lys Cys Ser Asn Ile Gln 805 810 815 Tyr Lys Asp Ser Gly Lys Glu Ile Tyr Thr Ile Glu Ile Pro Ile Ser 820 825 830 Ala Thr Lys Asn Val Pro Ser Asn Trp Val Gln Met Ala Ala Asn Lys 835 840 845 Thr Tyr Lys Arg Tyr Tyr Ser Asp Glu Val Arg Ala Leu Ala Arg Ser 850 855 860 Met Ala Glu Ala Lys Glu Ile His Lys Thr Leu Glu Glu Asp Leu Lys 865 870 875 880 Asn Arg Leu Cys Gln Lys Phe Asp Ala His Tyr Asn Thr Ile Trp Met 885 890 895 Pro Thr Ile Gln Ala Ile Ser Asn Ile Asp Cys Leu Leu Ala Ile Thr 900 905 910 Arg Thr Ser Glu Tyr Leu Gly Ala Pro Ser Cys Arg Pro Thr Ile Val 915 920 925 Asp Glu Val Asp Ser Lys Thr Asn Thr Gln Leu Asn Gly Phe Leu Lys 930 935 940 Phe Lys Ser Leu Arg His Pro Cys Phe Asn Leu Gly Ala Thr Thr Ala 945 950 955 960 Lys Asp Phe Ile Pro Asn Asp Ile Glu Leu Gly Lys Glu Gln Pro Arg 965 970 975 Leu Gly Leu Leu Thr Gly Ala Asn Ala Ala Gly Lys Ser Thr Ile Leu 980 985 990 Arg Met Ala Cys Ile Ala Val Ile Met Ala Gln Met Gly Cys Tyr Val 995 1000 1005 Pro Cys Glu Ser Ala Val Leu Thr Pro Ile Asp Arg Ile Met Thr Arg 1010 1015 1020 Leu Gly Ala Asn Asp Asn Ile Met Gln Gly Lys Ser Thr Phe Phe Val 1025 1030 1035 1040 Glu Leu Ala Glu Thr Lys Lys Ile Leu Asp Met Ala Thr Asn Arg Ser 1045 1050 1055 Leu Leu Val Val Asp Glu Leu Gly Arg Gly Gly Ser Ser Ser Asp Gly 1060 1065 1070 Phe Ala Ile Ala Glu Ser Val Leu His His Val Ala Thr His Ile Gln 1075 1080 1085 Ser Leu Gly Phe Phe Ala Thr His Tyr Gly Thr Leu Ala Ser Ser Phe 1090 1095 1100 Lys His His Pro Gln Val Arg Pro Leu Lys Met Ser Ile Leu Val Asp 1105 1110 1115 1120 Glu Ala Thr Arg Asn Val Thr Phe Leu Tyr Lys Met Leu Glu Gly Gln 1125 1130 1135 Ser Glu Gly Ser Phe Gly Met His Val Ala Ser Met Cys Gly Ile Ser 1140 1145 1150 Lys Glu Ile Ile Asp Asn Ala Gln Ile Ala Ala Asp Asn Leu Glu His 1155 1160 1165 Thr Ser Arg Leu Val Lys Glu Arg Asp Leu Ala Ala Asn Asn Leu Asn 1170 1175 1180 Gly Glu Val Val Ser Val Pro Gly Gly Leu Gln Ser Asp Phe Val Arg 1185 1190 1195 1200 Ile Ala Tyr Gly Asp Gly Leu Lys Asn Thr Lys Leu Gly Ser Gly Glu 1205 1210 1215 Gly Val Leu Asn Tyr Asp Trp Asn Ile Lys Arg Asn Val Leu Lys Ser 1220 1225 1230 Leu Phe Ser Ile Ile Asp Asp Leu Gln Ser 1235 1240

Claims

1-36. (canceled)

37. An isolated and purified nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having the amino acid sequence of AtMSH6 (SEQ ID NO:31).

38. The nucleic acid of claim 37 further comprising a regulation element operably linked to said AtMSH6-encoding sequence.

39. A plasmid or vector comprising the nucleic acid of claim 38.

40. A plant cell stably transformed, transfected or electroporated with the plasmid or vector of claim 39.

41. A plant comprising the cell of claim 40.

42. A process for at least partially inactivating the DNA mismatch repair system of a plant cell comprising:

transforming or transfecting said plant cell with a nucleic acid comprising a regulation element operably linked to a nucleotide sequence encoding a polypeptide having the amino acid sequence of AtMSH6 (SEQ ID NO:31);

growing said cell under conditions that permit expression of said AtMSH6-encoding sequence; and

hereby inactivating the DNA mismatch repair system of said plant cell.

43. The process of claim 42, wherein said plant is selected from the group consisting of Brassicaceae, Poaceae, Solanaceae, Asteraceae, Malvaceae, Fabaceae, Linaceae, Canabinaceae, Dauaceae, and Curcubitaceae.

44. A process for altering the mismatch repair system in a plant comprising introducing into said plant a chimeric gene expressing a polynucleotide which is capable of interfering with the expression of the nucleotide sequence encoding a AtMSH6 polypeptide (SEQ ID NO:31).

45. The process of claim 44, wherein said chimeric gene expresses a polynucleotide that is antisense with regard to the nucleic acid molecule comprising a nucleotide sequence encoding said AtMSH6 polypeptide.