Abstract:
The present invention relates to a plant, which is resistant to a pathogen of viral, bacterial, fungal or oomycete origin, wherein the plant has an increased homoserine level as compared to a plant that is not resistant to the said pathogen, in particular organisms of the phylum Oomycota. The invention further relates to a method for obtaining a plant, which is resistant to a pathogen of viral, bacterial, fungal or oomycete origin, comprising increasing the endogenous homoserine level in the plant.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a divisional application of U.S. application Ser. No. 12/092,253, which is a U.S. National Phase application filed under 35 U.S.C. §371 claiming priority to PCT Application Serial No. PCT/EP06/10535, filed Nov. 1, 2006, and PCT/EP06/10535 claims priority to PCT Application Serial No. PCT/EP/011718, filed Nov. 1, 2005, and is incorporated by reference herein. 
     
    
     BACKGROUND 
       [0002]    The present invention relates to disease resistant plants, in particular plants resistant to organisms of the phylum Oomycota, the oomycetes. The invention further relates to plant genes conferring disease resistance and methods of obtaining such disease resistant plants for providing protection to Oomycota pathogens. 
         [0003]    Resistance of plants to pathogens has been extensively studied, for both pathogen specific and broad resistance. In many cases resistance is specified by dominant genes for resistance. Many of these race-specific or gene-for-gene resistance genes have been identified that mediate pathogen recognition by directly or indirectly interacting with avirulence gene products or other molecules from the pathogen. This recognition leads to the activation of a wide range of plant defense responses that arrest pathogen growth. 
         [0004]    In plant breeding there is a constant struggle to identify new sources of mostly monogenic dominant resistance genes. In cultivars with newly introduced single resistance genes, protection from disease is often rapidly broken, because pathogens evolve and adapt at a high frequency and regain the ability to successfully infect the host plant. Therefore, the availability of new sources of disease resistance is highly needed. 
         [0005]    Alternative resistance mechanisms act for example through the modulation of the defense response in plants, such as the resistance mediated by the recessive m/o gene in barley to the powdery mildew pathogen  Blumeria graminis  f.sp.  hordei . Plants carrying mutated alleles of the wildtype MLO gene exhibit almost complete resistance coinciding with the abortion of attempted fungal penetration of the cell wall of single attacked epidermal cells. The wild type MLO gene thus acts as a negative regulator of the pathogen response. This is described in WO9804586. 
         [0006]    Other examples are the recessive powdery mildew resistance genes, found in a screen for loss of susceptibility to  Erysiphe cichoracearum . Three genes have been cloned so far, named PMR6, which encodes a pectate lyase-like protein, PMR4 which encodes a callose synthase, and PMR5 which encodes a protein of unknown function. Both m/o and pmr genes appear to specifically confer resistance to powdery mildew and not to oomycetes such as downy mildews. 
         [0007]    Broad pathogen resistance, or systemic forms of resistance such as SAR, has been obtained by two main ways. The first is by mutation of negative regulators of plant defense and cell death, such as in the cpr, lsd and acd mutants of  Arabidopsis . The second is by transgenic overexpression of inducers or regulators of plant defense, such as in NPR1 overexpressing plants. 
         [0008]    The disadvantage of these known resistance mechanisms is that, besides pathogen resistance, these plants often show detectable additional and undesirable phenotypes, such as stunted growth or the spontaneous formation of cell death. 
         [0009]    It is an object of the present invention to provide a form of resistance that is broad, durable and not associated with undesirable phenotypes. 
         [0010]    In the research that led to the present invention, an  Arabidopsis thaliana  mutant screen was performed for reduced susceptibility to the downy mildew pathogen  Hyaloperonospora parasitica . EMS-mutants were generated in the highly susceptible  Arabidopsis  line Ler eds1-2. Eight downy mildew resistant (dmr) mutants were analysed in detail, corresponding to 6 different loci. Microscopic analysis showed that in all mutants  H. parasitica  growth was severely reduced. Resistance of dmr3, dmr4 and dmr5 was associated with constitutive activation of plant defense. Furthermore, dmr3 and dmr4, but not dmr5, were also resistant to  Pseudomonas syringae  and  Golovinomyces orontii.    
         [0011]    In contrast, enhanced activation of plant defense was not observed in the dmr1, dmr2, and dmr6 mutants. The results of this research have been described in Van Damme et al. (2005) Molecular Plant-Microbe Interactions 18(6) 583-592. This article does however not disclose the identification and characterization of the DMR genes. 
       SUMMARY OF THE INVENTION 
       [0012]    According to the present invention it was now found that DMR1 is the gene encoding homoserine kinase (HSK). For  Arabidopsis  five different mutant dmr1 alleles have been sequenced each leading to a different amino acid change in the HSK protein. HSK is a key enzyme in the biosynthesis of the amino acids methionine, threonine and isoleucine and is therefore believed to be essential. The various dmr1 mutants show defects in HSK causing the plants to accumulate homoserine. The five different alleles show different levels of resistance that correlate to different levels of homoserine accumulation in the mutants. 
         [0013]    The present invention thus provides a plant, which is resistant to a pathogen of viral, bacterial, fungal or oomycete origin, characterized in that the plant has an altered homoserine level as compared to a plant that is not resistant to the said pathogen. 
         [0014]    This form of resistance is in particular effective against pathogens of the phylum Oomycota, such as  Albugo, Aphanomyces, Basidiophora, Bremia, Hyaloperonospora, Pachymetra, Paraperonospora, Perofascia, Peronophythora, Peronospora, Peronosclerospora, Phytium, Phytophthora, Plasmopara, Protobremia, Pseudoperonospora, Sclerospora, Viennotia  species. 
         [0015]    The resistance is based on an altered level of homoserine in planta. More in particular, the resistance is based on an increased level of homoserine in planta. Such increased levels can be achieved in various ways. 
         [0016]    First, homoserine can be provided by an external source. Second, the endogenous homoserine level can be increased. This can be achieved by lowering the enzymatic activity of the homoserine kinase gene which leads to a lower conversion of homoserine and thus an accumulation thereof. Alternatively, the expression of the homoserine kinase enzyme can be reduced. This also leads to a lower conversion of homoserine and thus an accumulation thereof. Another way to increase the endogenous homoserine level is by increasing its biosynthesis via the aspartate pathway. Reducing the expression of the homoserine kinase gene can in itself be achieved in various ways, either directly, such as by gene silencing, or indirectly by modifying the regulatory sequences thereof or by stimulating repression of the gene. 
         [0017]    Modulating the HSK gene to lower its activity or expression can be achieved at various levels. First, the endogenous gene can be directly mutated. This can be achieved by means of a mutagenic treatment. Alternatively, a modified HSK gene can be brought into the plant by means of transgenic techniques or by introgression, or the expression of HSK can be reduced at the regulatory level, for example by modifying the regulatory sequences or by gene silencing. 
         [0018]    In one embodiment of the invention, an increase (accumulation) in homoserine level in the plant is achieved by administration of homoserine to the plant. This is suitably done by treating plants with L-homoserine, e.g. by spraying or infiltrating with a homoserine solution. 
         [0019]    Treatment of a plant with exogenous homoserine is known from WO00/70016. This publication discloses how homoserine is applied to a plant resulting in an increase in the phenol concentration in the plant. The publication does not show that plants thus treated are resistant to pathogens. In fact, WO00/70016 does not disclose nor suggest that an increase in endogenous homoserine would lead to pathogen resistance. 
         [0020]    Alternatively, endogenous homoserine is increased by modulating plant amino acid biosynthetic or metabolic pathways. 
         [0021]    In one embodiment, the increased endogenous production is the result of a reduced endogenous HSK gene expression thus leading to a less efficient conversion of homoserine into phospho-homoserine and the subsequent biosynthesis of methionine and threonine. This reduced expression of HSK is for example the result of a mutation in the HSK gene leading to reduced mRNA or protein stability. 
         [0022]    In another embodiment reduced expression can be achieved by downregulation of the HSK gene expression either at the transcriptional or the translational level, e.g. by gene silencing or by mutations in the regulatory sequences that affect the expression of the HSK gene. An example of a method of achieving gene silencing is by means of RNAi. 
         [0023]    In a further embodiment the increase in endogenous homoserine level can be obtained by inducing changes in the biosynthesis or metabolism of homoserine. In a particular embodiment this is achieved by mutations in the HSK coding sequence that result in a HSK protein with a reduced enzymatic activity thus leading to a lower conversion of homoserine into phospho-homoserine. Another embodiment is the upregulation of genes in the aspartate pathway causing a higher production and thus accumulation of L-homoserine in planta. 
     
    
     
       DESCRIPTION OF DRAWINGS 
         [0024]      FIG. 1  shows orthologous HSK sequences that have been identified in publicly available databases and obtained by PCR amplification on cDNA and subsequent sequencing.  FIG. 1  shows the alignment of the amino acid sequences of the HSK proteins of  Arabidopsis thaliana  and orthologs from  Citrus sinensis, Populus trichocarpa  (1),  Populus trichocarpa  (2),  Solanum tuberosum  (2),  Vitis vinifera, Lactuca sativa, Solanum tuberosum  (1),  Solanum lycopersicum, Nicotiana benthamiana, Ipomoea nil, Glycine max, Phaseolus vulgaris, Cucumis sativus, Spinacia oleracea, Pinus taeda, Zea mays, and Oryza sativa  using the CLUSTAL W (1.82) multiple sequence alignment programme (EBI). Below the sequences the conserved amino acids are indicated by the dots, and the identical amino acids are indicated by the asterisks. The black triangles and corresponding text indicate the amino acids that are substituted in the five  Arabidopsis  dmr mutants [SEQ ID NOs. 39-47, 57, 88-89, 91-98, 100, 103, 110, 112, 144-151, 155, 158, 160, 165, 167, 205-211, 218, 225, 227, 264-271, 278, 280, 287, 324-331, 335, 347-348, 360, 364, 368, 370, 373, 375-376, 378, 380-381, and 384. 
           [0025]    Table 2 shows the Genbank accession numbers and GenInfo identifiers of the  Arabidopsis  HSK mRNA and orthologous sequences from other plant species. 
           [0026]      FIG. 2  shows the percentage of conidiophore formation by two  Hyaloperonospora parasitica  isolates, Cala2 and Waco9, on the mutants dmr1-1, dmr1-2, dmr1-3 and dmr1-4 and the parental line, Ler eds1-2, 7 days post inoculation. The conidiophores formed on the parental line were set to 100%. 
           [0027]      FIG. 3  is a graphic overview of the three major steps in the cloning of DMR1. a) Initial mapping of dmr1 resulted in positioning of the locus on the lower arm of chromosome 2 between positions 7.42 and 7.56 Mb. Three insert/deletion (INDEL) markers were designed (position of the markers F6P23, T23A1 and F5J6 is indicated by the black lines). These markers were used to identify recombinants from several 100 segregating F2 and F3 plants. Primer sequences of these INDEL markers and additional markers to identify the breakpoints in the collected recombinants is presented in table 3. b) One marker, At2g17270 (indicated by the grey line), showed the strongest linkage with resistance. The dmr1 locus could be further delimited to a region containing 8 genes, at2g17250-at2g17290. The eight genes were amplified and sequenced to look for mutations in the coding sequences using the primers described in table 4. DNA sequence analysis of all 8 candidate genes led to the discovery of point mutations in the At2g17265 gene in all 5 dmr1 mutants. c) Each dmr1 mutant has a point mutation at a different location in the At2g17265 gene, which encodes homoserine kinase. 
           [0028]      FIG. 4  shows a schematic drawing of the HSK coding sequence and the positions and nucleotide substitutions of the 5 different dmr1 mutations in the HSK coding sequence (the nucleotide positions, indicated by the black triangles, are relative to the ATG start codon which start on position 1). The 5′UTR and 3′UTR are shown by light grey boxes. Below the nucleotide sequence the protein sequence is shown. The HSK protein contains a putative transit sequence for chloroplast targeting (dark grey part). The amino acid changes resulting from the 5 dmr1 mutations are indicated at their amino acid (aa) position number (black triangles) in the HSK protein. 
           [0029]      FIG. 5  shows the position of the homoserine kinase enzyme in the aspartate pathway for the biosynthesis of the amino acids threonine, methionine and isoleucine. 
           [0030]      FIG. 6  shows the number of conidiophores per Ler eds1-2 seedlings 5 days post inoculation with two different isolates of  H. parasitica , Waco9 and Cala2. The inoculated seedlings were infiltrated with dH2O, D-homoserine (5 mM) or L-homoserine (5 mM) at 3 days post inoculation with the pathogen. Seedlings treated with L-homoserine show a complete absence of conidiophore formation and are thus resistant. 
           [0031]      FIG. 7  shows the growth and development of  H. parasitica  in seedlings treated with water, D-homoserine (5 mM), or L-homoserine (5 mM) as analysed by microscopy of trypan blue stained seedlings.
       a: Conidiophore formation after HS treatment on Ler eds1-2 seedlings (10× magnification). No conidiophore formation was detected after L-homoserine infiltration, whereas control plants show abundant sporulation.   b: Haustorial development is affected by L-homoserine (5 mM) infiltration (40× magnification), but not in plants treated with water or D-homoserine.       
 
           [0034]      FIGS. 8 and 9  show the nucleotide and amino acid sequence of the homoserine kinase gene (At2g17265, NM — 127281, GI:18398362) and protein (At2g17265, NP — 179318, GI:15227800) of  Arabidopsis thaliana , respectively [SEQ ID NOs. 99-100]. 
           [0035]      FIG. 10  shows the nucleotide and the predicted amino acid sequence of the homoserine kinase coding sequence (CDS) and protein, respectively, of  Lactuca sativa  [SEQ ID NOs. 101-102]. 
           [0036]      FIG. 11  shows the nucleotide and the predicted amino acid sequence of the homoserine kinase coding sequence (CDS) and protein, respectively, of  Vitis vinifera  [SEQ ID NOs. 103-104]. 
           [0037]      FIG. 12  shows the nucleotide and the predicted amino acid sequence of the homoserine kinase coding sequence (CDS) and protein, respectively, of  Cucumis sativus  [SEQ ID NOs. 105-106]. 
           [0038]      FIG. 13  shows the nucleotide and the predicted amino acid sequence of the homoserine kinase coding sequence (CDS) and protein, respectively, of  Spinacia oleracea  [SEQ ID NOs. 107-108]. 
           [0039]      FIG. 14  shows the nucleotide and the predicted amino acid sequence of the homoserine kinase coding sequence (CDS) and protein, respectively, of  Solanum lycopersicum  [SEQ ID NOs. 109-110]. 
       
    
    
     DETAILED DESCRIPTION 
       [0040]    This invention is based on research performed on resistance to  Hyaloperonospora parasitica  in  Arabidopsis  but is a general concept that can be more generally applied in plants, in particular in crop plants that are susceptible to infections with pathogens, such as Oomycota. 
         [0041]    The invention is suitable for a large number of plant diseases caused by oomycetes such as, but not limited to,  Bremia lactucae  on lettuce,  Peronospora farinosa  on spinach,  Pseudoperonospora cubensis  on members of the Cucurbitaceae family, e.g. cucumber,  Peronospora destructor  on onion,  Hyaloperonospora parasitica  on members of the Brasicaceae family, e.g. cabbage,  Plasmopara viticola  on grape,  Phytophthora infestans  on tomato and potato, and  Phytophthora sojae  on soybean. 
         [0042]    The homoserine level in these other plants can be increased with all techniques described above. However, when the modification of the HSK gene expression in a plant is to be achieved via genetic modification of the HSK gene or via the identification of mutations in the HSK gene, and the gene is not yet known it must first be identified. To generate pathogen-resistant plants, in particular crop plants, via genetic modification of the HSK gene or via the identification of mutations in the HSK gene, the orthologous HSK genes must be isolated from these plant species. Orthologs are defined as the genes or proteins from other organisms that have the same function. 
         [0043]    Various methods are available for the identification of orthologous sequences in other plants. 
         [0044]    A method for the identification of HSK orthologous sequences in a plant species, may for example comprise identification of homoserine kinase ESTs of the plant species in a database; designing primers for amplification of the complete homoserine kinase transcript or cDNA; performing amplification experiments with the primers to obtain the corresponding complete transcript or cDNA; and determining the nucleotide sequence of the transcript or cDNA. 
         [0045]    Suitable methods for amplifying the complete transcript or cDNA in situations where only part of the coding sequence is known are the advanced PCR techniques 5′RACE, 3′RACE, TAIL-PCR, RLM-RACE and vectorette PCR. 
         [0046]    Alternatively, if no nucleotide sequences are available for the plant species of interest, primers are designed on the HSK gene of a plant species closely related to the plant of interest, based on conserved domains as determined by multiple nucleotide sequence alignment, and used to PCR amplify the orthologous sequence. Such primers are suitably degenerate primers. 
         [0047]    Another reliable method to assess a given sequence as being a HSK ortholog is by identification of the reciprocal best hit. A candidate orthologous HSK sequence of a given plant species is identified as the best hit from DNA databases when searching with the  Arabidopsis  HSK protein or DNA sequence, or that of another plant species, using a Blast programme. The obtained candidate orthologous nucleotide sequence of the given plant species is used to search for homology to all  Arabidopsis  proteins present in the DNA databases (e.g. at NCBI or TAIR) using the BlastX search method. If the best hit and score is to the  Arabidopsis  HSK protein, the given DNA sequence can be described as being an ortholog, or orthologous sequence. 
         [0048]    HSK is encoded by a single gene in  Arabidopsis  and rice as deduced from the complete genome sequences that are publicly available for these plant species. In most other plant species tested so far, HSK appears to be encoded by a single gene, as determined by the analysis of mRNA sequences and EST data from public DNA databases, except for potato, tobacco and poplar for which two HSK homologs have been identified. The orthologous genes and proteins are identified in these plants by nucleotide and amino acid comparisons with the information that is present in public databases. 
         [0049]    Alternatively, if no DNA sequences are available for the desired plant species, orthologous sequences are isolated by heterologous hybridization using DNA probes of the HSK gene of  Arabidopsis  or another plant or by PCR methods, making use of conserved domains in the HSK coding sequence to define the primers. For many crop species, partial HSK mRNA sequences are available that can be used to design primers to subsequently PCR amplify the complete mRNA or genomic sequences for DNA sequence analysis. 
         [0050]    In a specific embodiment the ortholog is a gene of which the encoded protein shows at least 50% identity with the  Arabidopsis  HSK protein or that of other plant HSK proteins. In a more specific embodiment the homology is at least 55%, more specifically at least 60%, even more specifically at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%. 
         [0051]    After orthologous HSK sequences are identified, the complete nucleotide sequence of the regulatory and coding sequence of the gene is identified by standard molecular biological techniques. For this, genomic libraries of the plant species are screened by DNA hybridization or PCR with probes or primers derived from a known homoserine kinase gene, such as the above described probes and primers, to identify the genomic clones containing the HSK gene. Alternatively, advanced PCR methods, such as RNA Ligase Mediated RACE (RLM-RACE), can be used to directly amplify gene and cDNA sequences from genomic DNA or reverse-transcribed mRNA. DNA sequencing subsequently results in the characterization of the complete gene or coding sequence. 
         [0052]    Once the DNA sequence of the gene is known this information is used to prepare the means to modulate the expression of the homoserine kinase gene in any one of the ways described above. 
         [0053]    More in particular, to achieve a reduced HSK activity the expression of the HSK gene can be down-regulated or the enzymatic activity of the HSK protein can be reduced by amino acid substitutions resulting from nucleotide changes in the HSK coding sequence. 
         [0054]    In a particular embodiment of the invention, downregulation of HSK gene expression is achieved by gene-silencing using RNAi. For this, transgenic plants are generated expressing a HSK anti-sense construct, an optimized micro-RNA construct, an inverted repeat construct, or a combined sense-anti-sense construct, so as to generate dsRNA corresponding to HSK that leads to gene silencing. 
         [0055]    In an alternative embodiment, one or more regulators of the HSK gene are downregulated (in case of transcriptional activators) by RNAi. 
         [0056]    In another embodiment regulators are upregulated (in case of repressor proteins) by transgenic overexpression. Overexpression is achieved in a particular embodiment by expressing repressor proteins of the HSK gene from a strong promoter, e.g. the 35S promoter that is commonly used in plant biotechnology. 
         [0057]    The downregulation of the HSK gene can also be achieved by mutagenesis of the regulatory elements in the promoter, terminator region, or potential introns. Mutations in the HSK coding sequence in many cases lead to amino acid substitutions or premature stop codons that negatively affect the expression or activity of the encoded HSK enzyme. 
         [0058]    These and other mutations that affect expression of HSK are induced in plants by using mutagenic chemicals such as ethyl methane sulfonate (EMS), by irradiation of plant material with gamma rays or fast neutrons, or by other means. The resulting nucleotide changes are random, but in a large collection of mutagenized plants the mutations in the HSK gene can be readily identified by using the TILLING (Targeting Induced Local Lesions IN Genomes) method (McCallum et al. (2000) Targeted screening for induced mutations. Nat. Biotechnol. 18, 455-457, and Henikoff et al. (2004) TILLING. Traditional mutagenesis meets functional genomics. Plant Physiol. 135, 630-636). The principle of this method is based on the PCR amplification of the gene of interest from genomic DNA of a large collection of mutagenized plants in the M2 generation. By DNA sequencing or by looking for point mutations using a single-strand specific nuclease, such as the CEL-I nuclease (Till et al. (2004) Mismatch cleavage by single-strand specific nucleases. Nucleic Acids Res. 32, 2632-2641) the individual plants that have a mutation in the gene of interest are identified. 
         [0059]    By screening many plants, a large collection of mutant alleles is obtained, each giving a different effect on gene expression or enzyme activity. The gene expression or enzyme activity can be tested by analysis of HSK transcript levels (e.g. by RT-PCR), quantification of HSK protein levels with antibodies or by amino acid analysis, measuring homoserine accumulation as a result of reduced HSK activity. These methods are known to the person skilled in the art. 
         [0060]    The skilled person can use the usual pathogen tests to see if the homoserine accumulation is sufficient to induce pathogen resistance. 
         [0061]    Plants with the desired reduced HSK activity or expression are then back-crossed or crossed to other breeding lines to transfer only the desired new allele into the background of the crop wanted. 
         [0062]    The invention further relates to mutated HSK genes encoding HSK proteins with a reduced enzymatic activity. In a particular embodiment, the invention relates to the dmr1 alleles dmr1-1, dmr1-2, dmr1-3, dmr1-4 and dmr1-5. 
         [0063]    In another embodiment, the invention relates to mutated versions of the HSK genes of  Lactuca sativa, Vitis vinifera, Cucumis sativus, Spinacia oleracea  and  Solanum lycopersicum  as shown in  FIGS. 10-14  [SEQ ID NOs. 101-110]. 
         [0064]    The present invention demonstrates that plants having an increased homoserine level show resistance to pathogens, in particular of oomycete origin. With this knowledge the skilled person can actively modify the HSK gene by means of mutagenesis or transgenic approaches, but also identify so far unknown natural variants in a given plant species that accumulate homoserine or that have variants of the HSK gene that lead to an increase in homoserine, and to use these natural variants according to the invention. 
         [0065]    In the present application the terms “homoserine kinase” and “HSK” are used interchangeably. 
         [0066]    The present invention is illustrated in the following examples that are not intended to limit the invention in any way. In the examples reference is made to the following figures. 
       EXAMPLES 
     Example 1 
     Characterization of the Gene Responsible for Pathogen Resistance in Dmr Mutants 
       [0067]    Van Damme et al., 2005, supra disclose four mutants, dmr1-1, dmr1-2, dmr1-3 and dmr1-4 that are resistant to  H. parasitica . The level of resistance can be examined by counting conidiophores per seedling leaf seven day post inoculation with the  H. parasitica  Cala2 isolate (obtainable from Dr. E. Holub (Warwick HRI, Wellesbourne, UK or Dr. G. Van den Ackerveken, Department of Biology, University of Utrecht, Utrecht, NL). For the parental line, Ler eds1-2 (Parker et al., 1996, Plant Cell 8:2033-2046), which is highly susceptible, the number of conidiophores is set at 100%. The reduction in conidiophore formation on the infected dmr1 mutants compared to seedlings of the parental line is shown in  FIG. 2 . 
         [0068]    According to the invention, the gene responsible for resistance to  H. parasitica  in the dmr1 mutants of van Damme et al., 2005, supra has been cloned by a combination of mapping and sequencing of candidate genes. 
         [0069]    DMR1 was isolated by map-based cloning. The dmr1 mutants were crossed to the FN2 Col-0 mutant to generate a mapping population. The FN2 mutant is susceptible to the  H. parasitica  isolate Cala2, due to a fast neutron mutation in the RPP2A gene (Sinapidou et al., 2004, Plant J. 38:898-909). All 5 dmr1 mutants carry single recessive mutations as the F1 plants were susceptible, and approximately a quarter of the F2 plants displayed  H. parasitica  resistance. 
         [0070]    The DMR1 cloning procedure is illustrated in  FIG. 3  and described in more detail below. The map location of the dmr1 locus was first determined by genotyping 48 resistant F2 plants to be located on the lower arm of chromosome 2. From an additional screen for new recombinants on 650 F2 plants ˜90 F2 recombinant plants between two INDEL (insertion/deletion) markers on BAC T24112 at 7.2 Mb and BAC F5J6 at 7.56 Mb (according to the TIGR  Arabidopsis  genome release Version 5.0 of January 2004) were identified, which allowed to map the gene to a region containing a contig of 5 BACs. 
         [0071]    The F2 plants were genotyped and the F3 generation was phenotyped in order to fine map the dmr1 locus. The dmr1 mutation could be mapped to a ˜130 kb region (encompassing 3 overlapping BAC clones: F6P23, T23A1, and F5J6) between two INDEL markers located on BAC F6P23, at 7.42 Mb and F5J6 at 7.56 Mb (according to the TIGR  Arabidopsis  genome release Version 5.0 of January 2004). This resulted in an area of 30 putative gene candidates for the dmr1 locus, between 
         [0072]    the  Arabidopsis  genes with the TAIR codes AT2g17060 and AT2g17380. Additionally cleaved amplified polymorphic sequences (CAPS) markers were designed based on SNPs linked to genes AT2g17190, AT2g17200, AT2g17270, At2g17300, At2g17310 and At2g17360 genes. 
         [0073]    Analyses of 5 remaining recombinants in this region with these CAPS marker data left 8 candidate genes, At2g17230 (NM — 127277, GI:30679913), At2g17240 (NM — 127278, GI:30679916), At2g17250 (NM — 127279, GI:22325730), At2g17260 (NM — 127280, GI:30679922), At2g17265 (NM — 127281, GI:18398362), At2g17270 (NM — 127282, GI:30679927), At2g17280 (NM — 127283, GI:42569096), At2g17290 (NM — 127284, GI:30679934). Sequencing of all the 8 genes resulted in the finding of point mutations in the AT2g17265 coding gene in the five dmr1 alleles; dmr1-1, dmr1-2, dmr1-3, dmr1-4 and dmr1-5, clearly demonstrating that AT2g17265 is DMR1.  FIG. 3  shows a scheme of dmr1 with point mutations of different alleles. 
         [0074]    At2g17265 encodes the homoserine kinase (HSK) enzyme, so far the only  Arabidopsis  gene exhibiting this function. 
         [0075]    In  Arabidopsis , HSK is encoded by a single gene, At2g17265 (Lee &amp; Leustek, 1999, Arch. Biochem. Biophys. 372: 135-142). HSK is the fourth enzyme in the aspartate pathway required for the biosynthesis of the amino acids methionine, threonine and isoleucine. HSK catalyzes the phosphorylation of homoserine to homoserine phosphate ( FIG. 5 ). 
       Example 2 
     Amino Acid Analysis 
       [0076]    Homoserine phosphate is an intermediate in the production of methionine, isoleucine and threonine in  Arabidopsis . Since homoserine kinase has a key role in the production of amino acids, free amino acid levels were determined in the parental line Ler eds1-2 and the four different dmr1 mutants. For this amino acids from total leaves were extracted with 80% methanol, followed by a second extraction with 20% methanol. The combined extracts were dried and dissolved in water. After addition of the internal standard, S-amino-ethyl-cysteine (SAEC) amino acids were detected by automated ion-exchange chromatography with post column ninhydrin derivatization on a JOEL AminoTac JLC-500/V (Tokyo, Japan). 
         [0077]    Amino acid analysis of four different dmr1 mutants and the parental line, Ler eds1-2 showed an accumulation of homoserine in the dmr1 mutants, whereas this intermediate amino acid was not detectable in the parental line Ler eds1-2. There was no reduction in the level of methionine, isoleucine and threonine in the dmr1 mutants (Table 1). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Concentration (in pmol/mg fresh weight) of homoserine, methionine, 
               
               
                 threonine and isoleucine in above-ground parts of 2-week old seedlings 
               
               
                 of the parental line Ler eds1-2 and the mutants dmr1-1, dmr1-2, 
               
               
                 dmr1-3 and dmr1-4. 
               
             
          
           
               
                   
                 Homoserine 
                 Methionine 
                 Isoleucine 
                 Threonine 
               
               
                   
                   
               
             
          
           
               
                 dmr1-1 
                 964 
                 29 
                 12 
                 264 
               
               
                 dmr1-2 
                 7128 
                 14 
                 29 
                 368 
               
               
                 dmr1-3 
                 466 
                 11 
                 16 
                 212 
               
               
                 dmr1-4 
                 6597 
                 11 
                 32 
                 597 
               
               
                 Ler eds 1-2 
                 0 
                 7 
                 10 
                 185 
               
               
                   
               
             
          
         
       
     
         [0078]    Due to the reduced activity of the HSK in the dmr1 mutants, homoserine accumulates. This effect could be further enhanced by a stronger influx of aspartate into the pathway leading to an even higher level of homoserine. The high concentration of the substrate homoserine would still allow sufficient phosphorylation by the mutated HSK so that the levels of methionine, isoleucine and threonine are not reduced in the dmr1 mutants and the parental line, Ler eds1-2 (Table 1). 
       Example 3 
     Pathogen Resistance is Achieved by Application of L-Homoserine 
       [0079]    To test if the effect is specific for homoserine the stereo-isomer D-homoserine was tested. Whole seedlings were infiltrated with water, 5 mM D-homoserine and 5 mM L-homoserine. Only treatment with the natural amino acid L-homoserine resulted in resistance to  H. parasitica . Seedlings treated with water or D-homoserine did not show a large reduction in pathogen growth and were susceptible to  H. parasitica . The infiltration was applied to two  Arabidopsis  accessions, Ler eds1-2 and Ws eds1-1, susceptible to Cala2 and Waco9, respectively. Conidiophore formation was determined as an indicator for  H. parsitica  susceptibility. Conidiophores were counted 5 days post inoculation with  H. parasitica  and 2 days post infiltration with water, D-homoserine or L-homoserine. ( FIG. 6 ). L-homoserine infiltration clearly results in reduction of conidiophore formation and  H. parasitica  resistance. This was further confirmed by studying pathogen growth in planta by trypan blue staining of  Arabidopsis  seedlings. Plants were inoculated with isolate Cala2. Two days later the plants were treated by infiltration with water, 5 mM D-homoserine, and 5 mM L-homoserine. Symptoms were scored at 5 days post inoculation and clearly showed that only the L-homoserine-infiltrated seedlings showed a strongly reduced pathogen growth and no conidiophore formation ( FIG. 7 ). 
         [0080]    Microscopic analysis showed that only in L-homoserine treated leaves the haustoria, feeding structures that are made by  H. parasitica  during the infection process, are disturbed. Again it is shown that increased levels of homoserine in planta lead to pathogen resistance. 
       Example 4 
     Identification of HSK Orthologs in Crops 
     Screening of Libraries on the Basis of Sequence Homology 
       [0081]    The nucleotide and amino acid sequences of the homoserine kinase gene and protein of  Arabidopsis thaliana  are shown in  FIGS. 8 and 9  [SEQ ID NOs. 99-100]. 
         [0082]    Public libraries of nucleotide and amino acid sequences were compared with the sequences of  FIGS. 8 and 9  [SEQ ID NOs. 99-100]. 
         [0083]    This comparison resulted in identification of the complete HSK coding sequences and predicted amino acid sequences in  Citrus sinensis, Populus trichocarpa  (1),  Populus trichocarpa  (2),  Solanum tuberosum  (2),  Solanum tuberosum  (1),  Nicotiana benthamiana, Ipomoea nil, Glycine max, Phaseolus vulgaris, Pinus taeda, Zea mays , and  Oryza sativa . The sequence information of the orthologous proteins thus identified is given in  FIG. 1 . For many other plant species orthologous DNA fragments could be identified by BlastX as reciprocal best hits to the  Arabidopsis  or other plant HSK protein sequences. 
       Identification of Orthologs by Means of Heterologous Hybridisation 
       [0084]    The HSK DNA sequence of  Arabidopsis thaliana  as shown in  FIG. 8  [SEQ ID NO. 99] is used as a probe to search for homologous sequences by hybridization to DNA on any plant species using standard molecular biological methods. Using this method orthologous genes are detected by southern hybridization on restriction enzyme-digested DNA or by hybridization to genomic or cDNA libraries. These techniques are well known to the person skilled in the art. As an alternative probe the HSK DNA sequence of any other more closely related plant species can be used as a probe. 
       Identification of Orthologs by Means of PCR 
       [0085]    For many crop species, partial HSK mRNA or gene sequences are available that are used to design primers to subsequently PCR amplify the complete cDNA or genomic sequence. When 5′ and 3′ sequences are available the missing internal sequence is PCR amplified by a HSK specific 5′ forward primer and 3′ reverse primer. In cases where only 5′, internal or 3′ sequences are available, both forward and reverse primers are designed. In combination with available plasmid polylinker primers, inserts are amplified from genomic and cDNA libraries of the plant species of interest. In a similar way, missing 5′ or 3′ sequences are amplified by advanced PCR techniques; 5′RACE, 3′ RACE, TAIL-PCR, RLM-RACE or vectorette PCR. 
         [0086]    As an example the sequencing of the Lactuca sativa (lettuce) HSK cDNA is provided. From the Genbank EST database at NCBI several Lactuca HSK ESTs were identified using the tblastn tool starting with the  Arabidopsis  HSK amino acid sequence. Clustering and alignment of the ESTs resulted in a consensus sequence for a 5′ HSK fragment and one for a 3′ HSK fragment. To obtain the complete lettuce HSK cDNA the RLM-RACE kit (Ambion) was used on mRNA from lettuce seedlings. The 5′ mRNA sequence was obtained by using a primer that was designed in the 3′ HSK consensus sequence derived from ESTs (R1Sla: GCCTTCTTCACAGCATCCATTCC) [SEQ ID. NO 1] and the 5′ RACE primers from the kit. The 3′ cDNA sequence was obtained by using two primers designed on the 5′RACE fragment (Let3RACEOut: CCGTTGCGGTTAATGAGATT [SEQ ID NO. 2], and Let3RACEInn: TCGTGTTGGTGAATCCTGAA) [SEQ ID NO. 3] and the 3′ RACE primers from the kit. Based on the assembled sequence new primers were designed to amplify the complete HSK coding from cDNA to provide the nucleotide sequence and derived protein sequence as presented in  FIG. 10  [SEQ ID NOs. 101-102]. A similar approach was a used for  Solanum lycopersicum  ( FIG. 14  [SEQ ID NOs. 109-110]) and  Vitis vinifera  ( FIG. 11  [SEQ ID NOs. 103-104]). 
         [0087]    The complete HSK coding sequences from more than 10 different plants species have been identified from genomic and EST databases. From the alignment of the DNA sequences, conserved regions in the coding sequence were selected for the design of degenerate oligonucleotide primers (for the degenerate nucleotides the abbreviations are according to the IUB nucleotide symbols that are standard codes used by all companies synthesizing oligonucleotides; G=Guanine, A=Adenine, T=Thymine, C=Cytosine, R=A or G, Y=C or T, 
       M=A or C, K=G or T, S=C or G, W=A or T, B=C or G or T, D=G or A or T, H=A or C or T, V=A or C or G, N=A or C or G or T). 
       [0088]    The procedure for obtaining internal HSK cDNA sequences of a given plant species is as follows:
       1. mRNA is isolated using standard methods,   2. cDNA is synthesized using an oligo dT primer and standard methods,   3. using degenerate forward and reverse oligonucleotides a PCR reaction is carried out,   4. PCR fragments are separated by standard agarose gel electrophoresis and fragments of the expected size are isolated from the gel,   5. isolated PCR fragments are cloned in a plasmid vector using standard methods,   6. plasmids with correct insert sizes, as determined by PCR, are analyzed by DNA sequencing,   7. Sequence analysis using blastX reveals which fragments contain the correct internal HSK sequences,   8. The internal DNA sequence can then be used to design gene- and species-specific primers for 5′ and 3′ RACE to obtain the complete HSK coding sequence by RLM-RACE (as described above).       
 
         [0097]    As an example the sequencing of the Cucumis sativus (cucumber) HSK cDNA is provided. For cucumber two primer combinations were successful in amplifying a stretch of internal coding sequence from cDNA; combination 1: primer F1Kom (GAYTTCYTHGGMTGYGCCGT) [SEQ ID NO. 4] and M1 RC (GCRGCGATKCCRGCRCAGTT) [SEQ ID NO. 5], and combination 2: primer M1Kom (AACTGYGCYGGMATCGCYGC) [SEQ ID NO. 6] and R1Kom (CCATDCCVGGAATCAANGGVGC) [SEQ ID NO. 7]. After cloning and sequencing of the amplified fragments cucumber HSK-specific primers were designed for 5′ RACE (Cuc5RACEOut: AGAGGATTTTTACTAAGTTTATTCGTG [SEQ ID NO. 8] and Cuc5RACEInn: AGACATAATCTCCCAAGCCATCA [SEQ ID NO. 9]) and 3′ RACE (Cuc3RACEOut: TGATGGCTTGGGAGATTATGTCT [SEQ ID NO. 10] and Cuc3RACEInn: CACGAATAAACTTAGTAAAAATCCTCT [SEQ ID NO. 11). Finally the complete cucumber HSK cDNA sequence was amplified and sequenced ( FIG. 12  [SEQ ID NOs. 105-106]). A similar approach was a used for spinach,  Spinacia oleracea  ( FIG. 13  [SEQ ID NOs. 107-108]). 
         [0098]    Orthologs identified as described in this example can be modified using well-known techniques to induce mutations that reduce the HSK expression or activity. Alternatively, the genetic information of the orthologs can be used to design vehicles for gene silencing. All these sequences are then used to transform the corresponding crop plants to obtain plants that are resistant to Oomycota. 
       Example 5 
     Reduction of Homoserine Kinase Expression in  Arabidopsis  by Means of RNAi 
       [0099]    The production of HSK silenced lines has been achieved in  Arabidopsis  by RNAi. A construct containing two ˜750 bp fragments of the HSK exon in opposite directions was successfully transformed into the  Arabidopsis  Col-0 accession. The transformants were analysed for resistance to  H. parasitica , isolate Waco9. Several transgenic lines were obtained that confer resistance to  H. parasitica . Analysis of HSK expression and homoserine accumulation confirm that in the transformed lines the HSK gene is silenced, resulting in resistance to  H. parasitica.    
       Example 6 
     Mutation of Seeds 
       [0100]    Seeds of the plant species of interest are treated with a mutagen in order to introduce random point mutations in the genome. Mutated plants are grown to produce seeds and the next generation is screened for increased accumulation of homoserine. This is achieved by measuring levels of the amino acid homoserine, by monitoring the level of HSK gene expression, or by searching for missense mutations in the HSK gene by the TILLING method, by DNA sequencing, or by any other method to identify nucleotide changes. 
         [0101]    The selected plants are homozygous or are made homozygous by selfing or inter-crossing. The selected homozygous plants with increased homoserine levels are tested for increased resistance to the pathogen of interest to confirm the increased disease resistance. 
       Example 7 
     Transfer of a Mutated Allele into the Background of a Desired Crop 
       [0102]    Introgression of the desired mutant allele into a crop is achieved by crossing and genotypic screening of the mutant allele. This is a standard procedure in current-day marker assistant breeding of crops. 
       TABLES 
       [0103]    GI numbers (GenInfo identifier) and Genbank accession number for Expressed Sequence Tags (ESTs) and mRNA sequences of the  Arabidopsis  HSK mRNA and orthologous sequences from other plant species. 
         [0104]    A GI number (genInfo identifier, sometimes written in lower case, “gi”) is a unique integer which identifies a particular sequence. The GI number is a series of digits that are assigned consecutively to each sequence record processed by NCBI. The GI number will thus change every time the sequence changes. The NCBI assigns GI numbers to all sequences processed into Entrez, including nucleotide sequences from DDBJ/EMBL/Gen Bank, protein sequences from SWISS-PROT, PIR and many others. The GI number thus provides a unique sequence identifier which is independent of the database source that specifies an exact sequence. If a sequence in Gen Bank is modified, even by a single base pair, a new GI number is assigned to the updated sequence. The accession number stays the same. The GI number is always stable and retrievable. Thus, the reference to GI numbers in the table provides a clear and unambiguous identification of the corresponding sequence. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
                 Common 
                   
                   
                   
               
               
                 Species 
                 name 
                 Detail 
                 GI number 
                 Genbank 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 
                   Arabidopsis 
                 
                 Thale cress 
                 mRNA 
                 39104571 
                 AK117871 
               
               
                 
                   thaliana 
                 
               
               
                 
                   Citrus sinensis 
                 
                 Sweet Orange 
                 ESTs 
                 55935768 
                 CV886642 
               
               
                   
                   
                   
                 28618675 
                 CB293218 
               
               
                   
                   
                   
                 55935770 
                 CV886643 
               
               
                   
                   
                   
                 28619455 
                 CB293998 
               
               
                 
                   Glycine max 
                 
                 Soybean 
                 ESTs 
                 10846810 
                 BF069552 
               
               
                   
                   
                   
                 17401269 
                 BM178051 
               
               
                   
                   
                   
                 8283472 
                 BE021031 
               
               
                   
                   
                   
                 16348965 
                 BI974560 
               
               
                   
                   
                   
                 7285286 
                 AW597773 
               
               
                   
                   
                   
                 58024665 
                 CX711406 
               
               
                   
                   
                   
                 58017647 
                 CX704389 
               
               
                   
                   
                   
                 20449357 
                 BQ253481 
               
               
                   
                   
                   
                 16105339 
                 BI893079 
               
               
                   
                   
                   
                 37996979 
                 CF808568 
               
               
                   
                   
                   
                 37996460 
                 CF808049 
               
               
                   
                   
                   
                 6072786 
                 AW102173 
               
               
                   
                   
                   
                 26057235 
                 CA800149 
               
               
                   
                   
                   
                 6455775 
                 AW186458 
               
               
                   
                   
                   
                 6072724 
                 AW102111 
               
               
                   
                   
                   
                 9203587 
                 BE329811 
               
               
                 
                   Ipomoea nil 
                 
                 Japanese 
                 ESTs 
                 74407098 
                 CJ761918 
               
               
                   
                 morning glory 
                   
                 74402449 
                 CJ757269 
               
               
                   
                   
                   
                 74402115 
                 CJ756935 
               
               
                   
                   
                   
                 74388670 
                 CJ743490 
               
               
                 
                   Nicotiana 
                 
                 Tobacco 
                 ESTs 
                 39880685 
                 CK295868 
               
               
                 
                   benthamiana 
                 
                   
                   
                 39859026 
                 CK284950 
               
               
                   
                   
                   
                 39864851 
                 CK287885 
               
               
                   
                   
                   
                 39864855 
                 CK287887 
               
               
                   
                   
                   
                 39859024 
                 CK284949 
               
               
                   
                   
                   
                 39864853 
                 CK287886 
               
               
                   
                   
                   
                 39880683 
                 CK295867 
               
               
                   
                   
                   
                 39864849 
                 CK287884 
               
               
                 
                   Oryza sativa 
                 
                 Rice 
                 mRNA 
                 50916171 
                 XM_468550 
               
               
                   
                   
                   
                 32970537 
                 AK060519 
               
               
                 
                   Phaseolus 
                 
                 Common 
                 ESTs 
                 62708660 
                 CV535256 
               
               
                 
                   vulgaris 
                 
                 Bean 
                   
                 62710636 
                 CV537232 
               
               
                   
                   
                   
                 62708052 
                 CV534648 
               
               
                   
                   
                   
                 62709395 
                 CV535991 
               
               
                   
                   
                   
                 62710761 
                 CV537357 
               
               
                   
                   
                   
                 62708535 
                 CV535131 
               
               
                   
                   
                   
                 62708534 
                 CV535130 
               
               
                   
                   
                   
                 62711318 
                 CV537914 
               
               
                   
                   
                   
                 62707924 
                 CV534520 
               
               
                   
                   
                   
                 62710733 
                 CV537329 
               
               
                   
                   
                   
                 62709601 
                 CV536197 
               
               
                   
                   
                   
                 62709064 
                 CV535660 
               
               
                   
                   
                   
                 62708834 
                 CV535430 
               
               
                 
                   Pinus taeda 
                 
                 Loblolly Pine 
                 ESTs 
                 70780626 
                 DR690274 
               
               
                   
                   
                   
                 67490638 
                 DR092267 
               
               
                   
                   
                   
                 48933532 
                 CO162991 
               
               
                   
                   
                   
                 34354980 
                 CF396563 
               
               
                   
                   
                   
                 67706241 
                 DR117931 
               
               
                   
                   
                   
                 17243465 
                 BM158115 
               
               
                   
                   
                   
                 34349136 
                 CF390719 
               
               
                   
                   
                   
                 66981484 
                 DR057917 
               
               
                   
                   
                   
                 48932595 
                 CO162054 
               
               
                   
                   
                   
                 66689208 
                 DR011702 
               
               
                   
                   
                   
                 48933450 
                 CO162909 
               
               
                   
                   
                   
                 34350236 
                 CF391819 
               
               
                   
                   
                   
                 67706323 
                 DR118013 
               
               
                   
                   
                   
                 48932678 
                 CO162137 
               
               
                   
                   
                   
                 66981399 
                 DR057832 
               
               
                   
                   
                   
                 34354850 
                 CF396433 
               
             
          
           
               
                 
                   Populus 
                 
                 Poplar 
                 Genome v1.0, LG_IX, 
               
               
                   trichocarpa  1 
                   
                 149339-148242 
               
               
                   
                   
                 Expression confirmed by ESTs 
               
               
                 
                   Populus 
                 
                 Poplar 
                 Genome v1.0, scaffold_66, 
               
               
                   trichocarpa  2 
                   
                 1415935-1417032 
               
               
                   
                   
                 Expression confirmed by ESTs 
               
             
          
           
               
                 
                   Solanum 
                 
                 Potato 
                 ESTs 
                 66838966 
                 DR037071 
               
               
                   tuberosum  1 
                   
                   
                 61238361 
                 DN588007 
               
               
                   
                   
                   
                 39804315 
                 CK251362 
               
               
                   
                   
                   
                 39801776 
                 CK250065 
               
               
                   
                   
                   
                 9250052 
                 BE340521 
               
               
                   
                   
                   
                 39832341 
                 CK275363 
               
               
                   
                   
                   
                 21917848 
                 BQ116921 
               
               
                   
                   
                   
                 9249876 
                 BE340345 
               
               
                   
                   
                   
                 39815050 
                 CK258070 
               
               
                   
                   
                   
                 39804985 
                 CK251702 
               
               
                   
                   
                   
                 39804987 
                 CK251703 
               
               
                   
                   
                   
                 39825384 
                 CK268406 
               
               
                   
                   
                   
                 39832342 
                 CK275364 
               
               
                   
                   
                   
                 66838967 
                 DR037072 
               
               
                   
                   
                   
                 9250394 
                 BE340863 
               
               
                   
                   
                   
                 39804317 
                 CK251363 
               
               
                   
                   
                   
                 39825385 
                 CK268407 
               
               
                   
                   
                   
                 21375072 
                 BQ516203 
               
               
                 
                   Solanum 
                 
                 Potato 
                 ESTs 
                 39813353 
                 CK256373 
               
               
                   tuberosum  2 
                   
                   
                 39793361 
                 CK246131 
               
               
                   
                   
                   
                 39793359 
                 CK246130 
               
               
                   
                   
                   
                 39813352 
                 CK256372 
               
               
                 
                   Zea Mays 
                 
                 Maize 
                 ESTs 
                 76021237 
                 DT948407 
               
               
                   
                   
                   
                 76913306 
                 DV165065 
               
               
                   
                   
                   
                 71446162 
                 DR827212 
               
               
                   
                   
                   
                 71449720 
                 DR830770 
               
               
                   
                   
                   
                 78117576 
                 DV535963 
               
               
                   
                   
                   
                 91048486 
                 EB158904 
               
               
                   
                   
                   
                 71439095 
                 DR820145 
               
               
                   
                   
                   
                 76936546 
                 DV174774 
               
               
                   
                   
                   
                 76012246 
                 DT939416 
               
               
                   
                   
                   
                 78085419 
                 DV513812 
               
               
                   
                   
                   
                 71766843 
                 DR964780 
               
               
                   
                   
                   
                 76924795 
                 DV170131 
               
               
                   
                   
                   
                 71449067 
                 DR830117 
               
               
                   
                   
                   
                 91875652 
                 EB405609 
               
               
                   
                   
                   
                 71450175 
                 DR831225 
               
               
                   
                   
                   
                 78103551 
                 DV521979 
               
               
                   
                   
                   
                 78090555 
                 DV518929 
               
               
                   
                   
                   
                 78104654 
                 DV523072 
               
               
                   
                   
                   
                 76926251 
                 DV170768 
               
               
                   
                   
                   
                 78111568 
                 DV529965 
               
               
                   
                   
                   
                 71773353 
                 DR971257 
               
               
                   
                   
                   
                 71425952 
                 DR807002 
               
               
                   
                   
                   
                 93282458 
                 EB674722 
               
               
                   
                   
                   
                 78074199 
                 DV502633 
               
               
                   
                   
                   
                 76293328 
                 DV032896 
               
               
                   
                   
                   
                 78075462 
                 DV503896 
               
               
                   
                   
                   
                 91054750 
                 EB165168 
               
               
                   
                   
                   
                 86469295 
                 DY235665 
               
               
                   
                   
                   
                 74243218 
                 DT651132 
               
               
                   
                   
                   
                 74242899 
                 DT650813 
               
               
                   
                   
                   
                 101384764 
                 EB814428 
               
               
                   
                   
                   
                 91054750 
                 EB165168 
               
               
                   
                   
                   
                 71440426 
                 DR821476 
               
               
                   
                   
                   
                 78121780 
                 DV540164 
               
               
                   
                   
                   
                 78103550 
                 DV521978 
               
               
                   
                   
                   
                 86469294 
                 DY235664 
               
               
                   
                   
                   
                 91877777 
                 EB407734 
               
               
                   
                   
                   
                 67014441 
                 CO443190 
               
               
                   
                   
                   
                 76924794 
                 DV170130 
               
               
                   
                   
                   
                 76021236 
                 DT948406 
               
               
                   
                   
                   
                 71446161 
                 DR827211 
               
               
                   
                   
                   
                 78110960 
                 DV529358 
               
               
                   
                   
                   
                 78074736 
                 DV503170 
               
               
                   
                   
                   
                 71428043 
                 DR809093 
               
               
                   
                   
                   
                 86469052 
                 DY235422 
               
               
                   
                   
                   
                 71440425 
                 DR821475 
               
               
                   
                   
                   
                 78121779 
                 DV540163 
               
               
                   
                   
                   
                 78104653 
                 DV523071 
               
               
                   
                   
                   
                 37400920 
                 CF637820 
               
               
                   
                   
                   
                 78074198 
                 DV502632 
               
               
                   
                   
                   
                 71449719 
                 DR830769 
               
               
                 
                   Solanum 
                 
                 Tomato 
                   
                 58213736 
                 BP877213 
               
               
                 
                   lycopersicum 
                 
                   
                   
                 7333245 
                 AW621598 
               
               
                   
                   
                   
                 4386685 
                 AI482761 
               
             
          
           
               
                 Unigene 
                   
                 Sequence described in this patent 
               
               
                 SGN-U223239 
                   
                 application 
               
               
                 from Sol 
               
               
                 Genomics 
               
               
                 Network 
               
               
                 
                   Lactuca sativa 
                 
                 Lettuce 
                 Sequence described in this patent 
               
               
                   
                   
                 application 
               
               
                 
                   Vitis vinifera 
                 
                 Grape vine 
                 Sequence described in this patent 
               
               
                   
                   
                 application 
               
               
                 
                   Spinacia oleracea 
                 
                 Spinach 
                 Sequence described in this patent 
               
               
                   
                   
                 application 
               
               
                 
                   Cucumis sativus 
                 
                 Cucumber 
                 Sequence described in this patent 
               
               
                   
                   
                 application 
               
               
                   
               
             
          
         
       
     
       Table 3 
       [0105]    Primer sequences of insertion/deletion (INDEL, size difference indicated in brackets) markers and cleaved amplified polymorphics sequences (CAP, polymorphic restriction site indicated in brackets) used in the mapping of the DMR1 locus. 
         [0000]    
       
         
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 Primer 
                   
                   
                   
                   
                   
                   
               
               
                 name: BAC 
                   
                   
                   
                   
                   
                 GI 
               
               
                 and/or 
                   
                 SEQ 
                   
                   
                 TYPE 
                 number 
               
               
                 TAIR At 
                 Forward primer 
                 ID 
                 Reverse primer 
                 SEQ 
                 (size/ 
                 of TAIR At 
               
               
                 code 
                 sequence 
                 NO. 
                 sequence 
                 ID NO. 
                 enzyme) 
                 code 
               
               
                   
               
             
             
               
                 T24112 
                 AATCCAAATTT 
                 SEQ 
                 AAACGAAGAGTG 
                 SEQ 
                 INDEL 
                 18398180 
               
               
                   
                 CTT 
                 ID 
                 AC 
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 13 
                   
                   
               
               
                   
                   
                 12 
                   
                   
                   
                   
               
               
                   
               
               
                 (At2g16670) 
                 GCGAGAACAC 
                 SEQ 
                 AATGGTTGGAG 
                 SEQ 
                 (33) 
                   
               
               
                   
                 A 
                 ID 
                   
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 15 
                   
                   
               
               
                   
                   
                 14 
                   
                   
                   
                   
               
               
                   
               
               
                 F5J6 
                 CCGTCAGATC 
                 SEQ 
                 CAGAAGCTGATG 
                 SEQ 
                 INDEL 
                 23506018, 
               
               
                   
                 AGTC 
                 ID 
                 AT 
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 17 
                   
                   
               
               
                   
                   
                 16 
                   
                   
                   
                   
               
               
                   
               
               
                 (AT2g17370- 
                 CTCATCTTGTT 
                 SEQ 
                 CGTGGAAAGTA 
                 SEQ 
                 (30) 
                 30679966 
               
               
                 80) 
                   
                 ID 
                   
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 19 
                   
                   
               
               
                   
                   
                 18 
                   
                   
                   
                   
               
               
                   
               
               
                 F6P23 
                 CGGTTTCATGT 
                 SEQ 
                 AAGAAGAGAACT 
                 SEQ 
                 INDEL 
                 22325728 
               
               
                   
                 CGA 
                 ID 
                 GC 
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 21 
                   
                   
               
               
                   
                   
                 20 
                   
                   
                   
                   
               
               
                   
               
               
                 (AT2g17060) 
                 GGAAGATCAT 
                 SEQ 
                 GTCAACCTTCC 
                 SEQ 
                 (37) 
                   
               
               
                   
                 A 
                 ID 
                   
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 23 
                   
                   
               
               
                   
                   
                 22 
                   
                   
                   
                   
               
               
                   
               
               
                 T23A1 
                 TCCTTCCATGT 
                 SEQ 
                 AACAAATTTGCTT 
                 SEQ 
                 INDEL 
                 42570808, 
               
               
                   
                 CCG 
                 ID 
                 C 
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 25 
                   
                   
               
               
                   
                   
                 24 
                   
                   
                   
                   
               
               
                   
               
               
                 (AT2g17220- 
                 AAACCA 
                 SEQ 
                 CAGCCTTT 
                 SEQ 
                 (26) 
                 30679913 
               
               
                 30) 
                   
                 ID 
                   
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 27 
                   
                   
               
               
                   
                   
                 26 
                   
                   
                   
                   
               
               
                   
               
               
                 AT2g17190 
                 GAATAGAGGT 
                 SEQ 
                 CTCTTGTATGTTT 
                 SEQ 
                 CAP 
                 30679898 
               
               
                   
                 TGAT 
                 ID 
                 T 
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 29 
                   
                   
               
               
                   
                   
                 28 
                   
                   
                   
                   
               
               
                   
                 GGAAATCAAG 
                 SEQ 
                 ACTGGGCTGAT 
                 SEQ 
                 (MseI) 
                   
               
               
                   
                 A 
                 ID 
                   
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 31 
                   
                   
               
               
                   
                   
                 30 
                   
                   
                   
                   
               
               
                   
               
               
                 AT2g17200 
                 CCTCTCCACC 
                 SEQ 
                 CGATCCATTTCGT 
                 SEQ 
                 CAP 
                 30679902 
               
               
                   
                 CATT 
                 ID 
                 C 
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 33 
                   
                   
               
               
                   
                   
                 32 
                   
                   
                   
                   
               
               
                   
                 TCTAATTTCG 
                 SEQ 
                 AAGCAATCTAC 
                 SEQ 
                 (MboII) 
                   
               
               
                   
                   
                 ID 
                   
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 35 
                   
                   
               
               
                   
                   
                 34 
                   
                   
                   
                   
               
               
                   
               
               
                 AT2g17270 
                 GATGCAGCTA 
                 SEQ 
                 ACGAAAATATCAA 
                 SEQ 
                 CAP 
                 30679927 
               
               
                   
                 AATT 
                 ID 
                 A 
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 37 
                   
                   
               
               
                   
                   
                 36 
                   
                   
                   
                   
               
               
                   
                 ATCAGTGTGAA 
                 SEQ 
                 AAGCTCCTTC 
                 SEQ 
                 (NlaIII) 
                   
               
               
                   
                   
                 ID 
                   
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 39 
                   
                   
               
               
                   
                   
                 38 
                   
                   
                   
                   
               
               
                   
               
               
                 AT2g17300- 
                 AGGTAGGATG 
                 SEQ 
                 GCATGTTTTCTCT 
                 SEQ 
                 CAP 
                 30679937, 
               
               
                 05 
                 GTAT 
                 ID 
                 A 
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 41 
                   
                   
               
               
                   
                   
                 40 
                   
                   
                   
                   
               
               
                   
                 TATGTTTGAAC 
                 SEQ 
                 AGCGATAGAAG 
                 SEQ 
                 (EcoRI) 
                 22325732 
               
               
                   
                 T 
                 ID 
                   
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 43 
                   
                   
               
               
                   
                   
                 42 
                   
                   
                   
                   
               
               
                   
               
               
                 AT2g17310 
                 ATGGGTAACG 
                 SEQ 
                 CACATGTATAAG 
                 SEQ 
                 CAP 
                 42569097 
               
               
                   
                 AAAG 
                 ID 
                 GT 
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 45 
                   
                   
               
               
                   
                   
                 44 
                   
                   
                   
                   
               
               
                   
                 AGAGGATTAG 
                 SEQ 
                 CTTCCCATAGA 
                 SEQ 
                 (MseI) 
                   
               
               
                   
                 T 
                 ID 
                   
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 47 
                   
                   
               
               
                   
                   
                 46 
                   
                   
                   
                   
               
               
                   
               
               
                 AT2g17360 
                 CCAACAAGTAT 
                 SEQ 
                 CCACATCAAACTT 
                 SEQ 
                 CAP 
                 30679959 
               
               
                   
                 CCT 
                 ID 
                 A 
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 49 
                   
                   
               
               
                   
                   
                 48 
                   
                   
                   
                   
               
               
                   
                 CTTTTGTTGTT 
                 SEQ 
                 ATGAACTCCAC 
                 SEQ 
                 (MaeIII) 
                   
               
               
                   
                   
                 ID 
                   
                 ID NO. 
                   
                   
               
               
                   
                   
                 NO. 
                   
                 51 
                   
                   
               
               
                   
                   
                 50 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Primer sequences used for amplifying and sequencing of eight candidate DMR1 
               
               
                 genes for which the TAIR and GI codes are indicated 
               
             
          
           
               
                   
                   
                   
                 TAIR 
                   
               
               
                 Primer name 
                 Primer sequence 
                 SEQ ID NO. 
                 codes 
                 GI codes 
               
               
                   
               
               
                 MvD17230_F 
                 TTCCCGAAGTGTACATTAAAAGCT 
                 SEQ ID NO. 
                 At2g17230 
                 30679913 
               
               
                   
                 C 
                 52 
                   
                   
               
               
                   
               
               
                 MvD17230_R 
                 TATGTCATCCCCAAGAGAAGAAG 
                 SEQ ID NO. 
                 At2g17230 
                 30679913 
               
               
                   
                 AC 
                 53 
                   
                   
               
               
                   
               
               
                 MvD17240-F 
                 CAATAAAAGCCTTTAAAAGCCCAC 
                 SEQ ID NO. 
                 At2g17240 
                 30679916 
               
               
                   
                 T 
                 54 
                   
                   
               
               
                   
               
               
                 MvD17240_R 
                 TAGCTTCTGAAACTGTGGCATTAC 
                 SEQ ID NO. 
                 At2g17240 
                 30679916 
               
               
                   
                 A 
                 55 
                   
                   
               
               
                   
               
               
                 MvD17250_1F 
                 CATGATTTGAGGGGTATATCCAAA 
                 SEQ ID NO. 
                 At2g17250 
                 22325730 
               
               
                   
                 A 
                 56 
                   
                   
               
               
                   
               
               
                 MvD17250_1R 
                 GGAGGTGGGATTTGAGATAAAAC 
                 SEQ ID NO. 
                 At2g17250 
                 22325730 
               
               
                   
                 TT 
                 57 
                   
                   
               
               
                   
               
               
                 MvD17250_2F 
                 TAGCCTAGAACTCTCTGTTCGCAA 
                 SEQ ID NO. 
                 At2g17250 
                 22325730 
               
               
                   
                 G 
                 58 
                   
                   
               
               
                   
               
               
                 MvD17250_2R 
                 CATTATTTTGCGTAGTTGTGAGTG 
                 SEQ ID NO. 
                 At2g17250 
                 22325730 
               
               
                   
                 G 
                 59 
                   
                   
               
               
                   
               
               
                 MvD17250_3F 
                 CGAAGAAATCCTACAATCAACCAT 
                 SEQ ID NO. 
                 At2g17250 
                 22325730 
               
               
                   
                 C 
                 60 
                   
                   
               
               
                   
               
               
                 MvD17250_3R 
                 TCTCACAATTCCCATCTCTTACTC 
                 SEQ ID NO. 
                 At2g17250 
                 22325730 
               
               
                   
                 C 
                 61 
                   
                   
               
               
                   
               
               
                 MvD17260_1F 
                 TTACTCATTTGGGTGAACAGAACA 
                 SEQ ID NO. 
                 At2g17260 
                 30679922 
               
               
                   
                 A 
                 62 
                   
                   
               
               
                   
               
               
                 MvD17260_1R 
                 ATCATCCCTAATCTCTCTGCTTCC 
                 SEQ ID NO. 
                 At2g17260 
                 30679922 
               
               
                   
                 T 
                 63 
                   
                   
               
               
                   
               
               
                 MvD17260_2F 
                 GATTAAGATACGGGGAATGGAGT 
                 SEQ ID NO. 
                 At2g17260 
                 30679922 
               
               
                   
                 CT 
                 64 
                   
                   
               
               
                   
               
               
                 MvD17260_2R 
                 ATGCAGACAAATAAGATGGCTCTT 
                 SEQ ID NO. 
                 At2g17260 
                 30679922 
               
               
                   
                 G 
                 65 
                   
                   
               
               
                   
               
               
                 MvD17260_3F 
                 GTTGTTGCTCCTGTCACAAGACTT 
                 SEQ ID NO. 
                 At2g17260 
                 30679922 
               
               
                   
                 A 
                 66 
                   
                   
               
               
                   
               
               
                 MvD17260_3R 
                 GAACAAAGACGAAGCCTTTAAAC 
                 SEQ ID NO. 
                 At2g17260 
                 30679922 
               
               
                   
                 AA 
                 67 
                   
                   
               
               
                   
               
               
                 MvD17265_F 
                 GAGGACTGCATCTAGAAGACCCA 
                 SEQ ID NO. 
                 At2g17265 
                 18398362 
               
               
                   
                 TA 
                 68 
                   
                   
               
               
                   
               
               
                 MvD17265_R 
                 TGGGCTCTCAACTATAAAGTTTGC 
                 SEQ ID NO. 
                 At2g17265 
                 18398362 
               
               
                   
                 T 
                 69 
                   
                   
               
               
                   
               
               
                 MvD17270_F1 
                 TAACGGTAAAGCAACGAATCTATC 
                 SEQ ID NO. 
                 At2g17270 
                 30679927 
               
               
                   
                 C 
                 70 
                   
                   
               
               
                   
               
               
                 MvD17270_R1 
                 TCAAACTGATAACGAGAGACGTT 
                 SEQ ID NO. 
                 At2g17270 
                 30679927 
               
               
                   
                 GA 
                 71 
                   
                   
               
               
                   
               
               
                 MvD17270_F2 
                 TTGCGTTCGTTTTTGAGTCTTTTA 
                 SEQ ID NO. 
                 At2g17270 
                 30679927 
               
               
                   
                 T 
                 72 
                   
                   
               
               
                   
               
               
                 MvD17270_R2 
                 AAACCAGACTCATTCCTTTGACAT 
                 SEQ ID NO. 
                 At2g17270 
                 30679927 
               
               
                   
                 C 
                 73 
                   
                   
               
               
                   
               
               
                 MvD17280_F1 
                 TTTAGGATCTCTGCCTTTTCTCAA 
                 SEQ ID NO. 
                 At2g17280 
                 42569096 
               
               
                   
                 C 
                 74 
                   
                   
               
               
                   
               
               
                 MvD17280_R1 
                 GAGAAATCAATAGCGGGAAAGAG 
                 SEQ ID NO. 
                 At2g17280 
                 42569096 
               
               
                   
                 AG 
                 75 
                   
                   
               
               
                   
               
               
                 MvD17280_F2 
                 GCTTAAATAGTCCTCCTTTCCTTG 
                 SEQ ID NO. 
                 At2g17280 
                 42569096 
               
               
                   
                 C 
                 76 
                   
                   
               
               
                   
               
               
                 MvD17280_R2 
                 TCTGCTGGTTCTCATGTTGATAGA 
                 SEQ ID NO. 
                 At2g17280 
                 42569096 
               
               
                   
                 G 
                 77 
                   
                   
               
               
                   
               
               
                 MvD17290_F1 
                 CTCTCCTTCATCATTTCACAAATC 
                 SEQ ID NO. 
                 At2g17290 
                 30679934 
               
               
                   
                 C 
                 78 
                   
                   
               
               
                   
               
               
                 MvD17290_R1 
                 TTCCTCTCGCTGTAATGACCTCTA 
                 SEQ ID NO. 
                 At2g17290 
                 30679934 
               
               
                   
                 T 
                 79 
                   
                   
               
               
                   
               
               
                 MvD17290_F2 
                 TGCCACAGGTGTTGACTATGC 
                 SEQ ID NO. 
                 At2g17290 
                 30679934 
               
               
                   
                   
                 80 
                   
                   
               
               
                   
               
               
                 MvD17290_R2 
                 TGCTCTTAAACCCGCAATCTC 
                 SEQ ID NO. 
                 At2g17290 
                 30679934 
               
               
                   
                   
                 81 
                   
                   
               
               
                   
               
               
                 MvD17290_F3 
                 GAAGATGGCTTTAAAGGTCAGTTT 
                 SEQ ID NO. 
                 At2g17290 
                 30679934 
               
               
                   
                 GT 
                 82 
                   
                   
               
               
                   
               
               
                 MvD17290_R3 
                 AGCAACAACAACTAAAAGGTGGA 
                 SEQ ID NO. 
                 At2g17290 
                 30679934 
               
               
                   
                 AG 
                 83