PL187851B1 - Gene giving immunity from diseases among plants and application thereof - Google Patents

Abstract

The invention concerns the location and characterization of a gene (designated NIM1) which is a key component of the SAR pathway and which in connection with chemical and biological inducers enables induction of SAR gene expression and broad spectrum disease resistance to plants. The invention further concerns plants transformed with the NIM1 gene as well as methods employing the gene to create the transgenic plants and employing the gene in a screening assay for compounds capable of inducing broad spectrum disease resistance in plants.

Description

The invention relates to an isolated DNA molecule capable of conferring disease resistance on plants, and an isolated DNA molecule capable of conferring universal disease susceptibility to plants, as well as a plasmid, a chimeric gene, a recombinant vector, a plant expression cassette, and a plant cell in which these isolated molecules are used. . The invention also relates to the use of an isolated DNA molecule and the use of resistant plant cells.

In particular, the invention relates to the identification, isolation and characterization of a gene associated with a broad spectrum of disease resistance in plants and the modification of the gene.

Plants are constantly attacked by a wide range of pathogenic organisms, including viruses, fungi and nematodes. Crop plants are particularly vulnerable to these attacks, as they are generally bred as large, genetically homogeneous monocultures, and when disease attack occurs, losses occur to the entire crop population.

Most plants have their own innate defense mechanisms against pathogenic organisms. Natural variation in resistance to plant pathogens has been and is identified by plant breeders and pathologists and is being crossbreed into many breeding plants. These natural resistance genes often provide high levels of resistance or resistance to pathogens.

In many plant species, initial immunization with a necrotizing pathogen can immunize the plant against subsequent infection. This acquired disease resistance was first documented in 1901 and is believed to play an important role in plant protection in nature. Particularly well characterized phenomena of resistance in plants are the phenomenon of systemic acquired resistance (SAR) and induced resistance in plants such as tobacco, Arabidopsis and cucumber. In these systems, inoculation by a necrotizing pathogen provides systemic protection against subsequent infections with this pathogen and certain other agricultural, bacterial, fungal and viral pathogens.

Systemic acquired resistance can also be induced by chemical immunizing compounds, some chemicals that induce an immune response in plants. Such compounds may be of natural origin such as salicylic acid (SA) or they may be synthetic compounds such as 2,6 dichloroisonicotinic acid (NA) and benzo (1,2,3) thiadizol-7-carbothioic acid methyl ester (BTH) . Treatment with the pathogen or the immunizing compound induces the expression of at least nine sets of genes in tobacco, the best characterized species. Various numbers and types of genes may be expressed in other plants. The level of induction for SAR related genes induced by the immunizing compounds is up to 10,000 times above background. SAR is characterized by the expression of SAR genes, including pathogenesis related (PR) genes.

SAR genes are induced after infection with a pathogen. Some of these genes play a role in providing systemic acquired resistance in the plant. These plant proteins are induced in large amounts in response to infection by a variety of pathogens including viruses, bacteria and fungi. PR proteins were first detected in tobacco plants (Nicotiana tabacum) hypersensitive to tobacco mosaic virus (TMV) infection. PR proteins were then found in a number of plant species (see Redolfi et al. (1983) Neth. J. Plant Pathol. 89: 245254; Van Loon (1985) Plant Mol. Biol. 4: 111-116; and Uknes et al. (1992) Plant Cell 4: 645-656 Such proteins are believed to be a common plant defense system against infection by pathogens.

Pathogenesis-related proteins include, but are not limited to, the SAR8.2a and SAR8.2b proteins, the acidic and basic forms of tobacco PR-1a, Pr-1b, and PR-1c; major proteins PR-1 ', PR-2, PR-2', PR-2, PR-N, PR-O, PR-O ', PR-4, PR-P, PR-Q, PR-S and PR -R; cucumber peroxidase; cucumber essential peroxidase; chitinase, which is the principal counterpart of PR-P or PR-q; beta-1,3-glucanase (glucano endo 1,3-beta glucosidase, E.C 3.2.1.30) which is essentially the counterpart of PR-2, PR-N or PR-O; and pathogen inducible cucumber chitinase. Such proteins are disclosed, for example, in Uknes et al. (1982) The Plant Cell 4: 645665 and references cited therein.

SAR or SAR-like genes are expressed in all plant species showing acquired systemic resistance. The expression of such genes can be determined

187,851 by probing with known SAR DNA sequences. For example, see Lawton et al. (1992) Proceedings of the Second European Federation of Plant Pathology (1983), in Mechanisms of Defense Responses in Plants, B. Fritig and M. Legrand (eds), Kluwer Academic Publishers, Dordrecht, pp. 410-420; Uknes et al. (1992) The Plant Cell 4: 645-656; and Ward et al. (1991) The Plant Cell 3: 1085-1094. Hybridization and cloning methods are well known in the art. See, for example, Molecular Cloning, A Laboratory Manual, 2nd ed. 2, Volumes 1-3, Sambrook et al. (Eds), Cold Spring Harbor Laboratory Press (1979) and references cited therein.

Alternatively, such SAR or SAR-like genes can be found using other methods such as protein screening, +/- searches, etc. See, for example, Liang and Pardee (1992) Science 257: 987-971 and St. John and Davis (1979) Cell 16: 443.

Despite extensive research and the use of advanced and intensive methods of crop protection, including genetic transformation in plants, disease losses still amount to billions of dollars annually. Disease resistance genes have been cloned previously, but transgenic plants transformed with these genes would typically be resistant to only a fraction of the strains of a given pathogen species. Despite attempts to clone genes related to SAR, the gene controlling a wide spectrum of diseases has not been isolated and characterized so far.

Several lines of evidence indicate that endogenously produced SA is involved in the linking signal transduction pathway linking the perception of pathogen infection to the onset of SAR. Mutants that retain the ability to accumulate SA in response to a pathogen but have lost the ability to induce SAR or resistance genes after SA or INA use have been described by Delaney et al., Proc. Natl. Acad. Sci. 92: 6602-6606 (1995) and WO94 / 16077, which is hereby incorporated by reference in its entirety.

These mutants have now been found to contain a mutant gene, which in its wild-type gene controls SAR expression and the SAR itself. The present invention recognizes that the mutant gene renders mutant plants susceptible to a wide range of diseases and makes them non-inducible by pathogens and chemical inducers.

The present invention relates to the identification, isolation and characterization of a wild gene (NIMI), a gene that allows SAR to be activated in a plant and the expression of SAR genes in response to biological and chemical inducers.

A mutant gene has been identified in mutagenized Arabidopsis plants. These plants have been found to be defective in their normal responses to pathogen infection in that they do not express genes associated with systemic acquired resistance (SAR), nor are they capable of showing SAR. These mutants contain a defective gene that has been marked with them (noninducible immunity).

The present invention also relates to the use of the cloned NIMI gene and variants thereof to create transgenic plants which have broad resistance to disease, and the transgenic plants thus produced. The invention further relates to the use of the cloned NIMI gene and variants thereof in a screening method to identify compounds capable of inducing broad-spectrum resistance in plants.

Brief description of the drawings.

Figure 1 shows the effect of chemical inducers on the expression of the PR gene in wild type and n1 plants.

Figure 2 - PR-1 gene expression in Ws-0 plants infected with the pathogen and therewith for 6 days after the onset of infection.

Figure 3 - SA accumulation levels in Ws-0 plants infected with P. syringae and therewith.

Figure 4 - Genetic map of the NIMI region as determined by AFLP and SSLP analysis.

Figure 5 - Physical map of the NIMI region as determined by YAC clone analysis.

Figure 6 - Physical map of the large PlBAC contig.

Figure 7 - Physical map showing the position of the P1 and BAC clones with respect to the flanking AFLP and YAC markers.

Figure 8 - Physical map of a further extended P1 / BAC contig containing the NIM 1 gene.

Figure 9 - Integrated genetic map and detailed physical map of the NIM region.

Figure 10 - Integrated map of the NIMI region.

Figure 11 - Integrated map of the NIMI region including new AFLP markers.

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Figure 12 is a schematic representation of the D169JC105 recombinants.

Figure 13 - Global map of the chromosomal area surrounding NIM1 with recombinants indicated, including BACi, YACi and cosmids in the NIM1 region.

Figure 14 - Provides the sequence of the 9.9 kb region of the BAC-04 clone containing the NIM1 gene. Figure 15 shows the NIM1 nucleic acid sequence and the amino acid sequence of the NIM1 gene product, including changes in the various alleles.

Figure 16 - NIM1 expression induced by INA, BTH, SA and pathogen in wild-type and mutant nim1 alleles.

Figure 17 - PR-1 expression in nim1 mutants and human type plants.

Figure 18 - Disease resistance in various nim 1 mutants.

Figure 19 is a comparison of the amino acid sequences of the NIM1 protein Expression Label regions and the cDNA protein products 4 of the rice gene sequence (SEQ ID NO: 3).

Definitions: AA: amino acid AFLP: Length polymorphism of amplified fragments avrRpt2: Rpt2 avirulence gene, isolated from Pseudomonas syringae BAC: Artificial bacterial chromosome BTH: Benzo (1,2,3) -thiadiazole-7-carbothioic acid, S-methyl ester Col: Arabidopsis ecotype Columbia Ecs: Combinations of enzymes INA: 2,6-dichloroisonicotinic acid Ler: Arabidopsis ecotype Landsberg erecta NIM1: a wild-type gene which confers resistance to a disease in a plant him: a mutant NIM1 allele, making the plant susceptible to disease him 1: mutated plant line ORF: open reading frame Pcs: primer combinations ARE: salicylic acid SAR: acquired systemic resistance SSLP: Length polymorphism of simple sequences In so: Arabidopsis ecotype Wassilewskija YAC: Artificial Yeast Chromosome

The NIM1 gene was cloned by mapping and walking techniques which indicate that the gene is contained in the region of approximately 105 kb (See figure 13 and table 16). This area is bounded by marker L84.6b on the left and marker L84.T2 on the right. Only three overlapping cosmids made of the wild-type DNA in the 105 kb region complement the phenotype of the nim1 mutant (Figure 13 and Table 16). These three cosmids share a common area only in area

9.9 kb defined the left end of cosmid clone D7 and extends the ^7th end of cosmid D5 as shown in Figure 13. Many other cosmids captured in other regions of the 105 kb region did not complement the nim1 phenotype (Figure 13 and Table 16). An almost full-length cDNA clone for the NIM1 gene points to the appropriate intron-exon boundaries and defines the amino acid sequence of the gene product. Only the region of the NIM1 gene within the 9.9 kb complementary region has sequence changes in the different mutant alleles thereof (Table 18). Three other potential gene regions showed no sequence changes that are associated with the nim1 phenotype. The sequence changes found in the region of the NIM1 gene are consistent with altered function or loss of function of the gene product. The severity of a change in the NIM1 gene region in a given mutant allele is roughly correlated with the observed physiological severity of the nim1 allele. Only the NIM1 gene region had detectable (transcription) RNA and this RNA showed numerous changes consistent with the physiological role of NIM1 in pathogenesis (Table 18 and Figure 16).

The present invention relates to an isolated gene fragment, the NIM1 gene, which is a key component of the systemic acquired resistance (SAR) pathway in plants. The NIM1 gene is involved in the activation of SAR by chemical and biological inducers and, in combination with such inducers, is required for SAR and SAR gene expression.

187 851

The location of the SAR gene is determined by molecular biology analysis of the genome of mutant plants known to carry a mutant gene with them, rendering host plants extremely sensitive to a wide range of pathogens and rendering them incapable of responding to pathogens and chemical SAR inducers.

Their mutants are useful as "universally susceptible to disease" (UDS) plants because they are susceptible to many strains and phenotypes of host plant pathogens and also to pathogens that do not normally infect the host plant but which infect other hosts . They can be generated by treating seeds or other biological materials with mutagenic agents and then selecting progeny plants with the UDS phenotype by treating them with known chemical inducers (e.g., INA) of a systemic resistance response and then infecting the plant with a known pathogen. Non-inducible mutants develop acute signs of disease under these conditions, while non-mutant plants are induced by the chemical into systemic acquired resistance. N and m mutants may also be selected from mutant populations obtained by chemical and post-irradiation mutagenesis as well as from populations generated by T-DNA insertion and transposon-induced mutagenesis.

Techniques for generating mutant plant lines are well known in the art. The nim plant phenotype is used as a tool to identify the isolated gene fragment that allows the expression of resistance to a wide range of diseases in plants.

An isolated DNA molecule comprising a mutant NlMl gene which is the gene thereof is encompassed by the present invention.

When the mutant or plant is used therein to isolate the wild-type NlMl gene necessary for constitutive expression of SAR genes, the resistance trait, in combination with other production and quality important traits, can be incorporated into a plant line by breeding. Culture approaches and techniques are known in the art. See, for example, Welsh J.R., Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, NY (1981); Crop Breeding, Wood D.R. (eds.), American Society of Agronomy, Madison, Wisconsin (1983); Mayo, O., The Theory of Plant Breeding, Second Edition, Clarendon Press, Oxford (1987); Singh, D.P., Breeding for Resistance to Diseases and Insect Pests, Springer-Verlag, NY (1986); and Wricke and Weber, Quantitative Genetics and Selection Plant Breeding, Walter de Gruyter and Co., Berlin (1986).

A further object of the invention is a hybrid gene containing a plant active promoter operably linked to a heterologous DNA molecule encoding the amino acid sequence of the NlMl gene product and variants thereof according to the invention.

Methodologies for the construction of plant expression cassettes as well as for the introduction of foreign DNA into plants are generally described in the art. Generally, Ti vectors have been used to introduce foreign DNA into plants. Direct DNA uptake, liposomes, electroporation, microinjection and microprojectiles have also been used for introduction. Such methods have been published in the art. See, for example, Bilang et al. (1991) Gene 100: 247-250; Scheid et al. (1991) Mol. Gen. Genet. 228: 104-112; Guerche et al. (1987) Plant Science 52: 111-116; Neuhause et al. (1987) Theor. Appl. Genet. 75: 30-36; Klein et al. (1987) Nature 327: 70-73; Howell et al. (1980) Science 208: 1265; Horsch et al. (1985) Science 227: 1229-1231; DeBlock et al. (1989) Plant Physiology 91: 694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds) Academic Press, lnc. (1988): and Methods in Plant Molecular Biology (Schuler and Zielinski, eds) Academic Press, lnc. (1989). See also US Patent Application Serial No. 08 / 438,666 filed May 10, 1995, and WO 93/07278, both of which are herein incorporated by reference in their entirety. It is understood that the method of transformation will depend on the plant cell to be transformed.

It is also recognized that the components of the expression cassette may be modified to increase expression. For example, truncated sequences, nucleotide substitutions, or other modifications may be used. Plant cells transformed with such a modified expression system would over-express or constitutively express the SAr genes necessary for SAR activation.

A DNA molecule or gene fragment conferring disease resistance to plants by allowing the expression of SAR genes can be incorporated into a bacterial or plant cell using

Conventional Recombinant DNA Technology. Generally, this includes insertion of the DNA molecule into an expression system in which the DNA molecule is heterologous (ie, is not normally present). The heterologous DNA molecule is inserted into the expression system or vector in the correct orientation and reading frame. The vector contains the elements needed for the transcription and translation of inserted protein-coding sequences. A large number of vector systems known in the art, such as plasmids, can be used. bacteriophages and other modified viruses. Suitable vectors include, but are not limited to, viral vectors such as the lambda vector system Igt11, Igt10, and Charon 4; plasmid vectors such as pBI121, pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKClOl, pCDNAII; and other similar systems. DNA sequences can be cloned into the vector using industry standard procedures as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory, Cold Spring Harbor, New York (1982).

A further object of the invention is a recombinant vector containing the hybrid gene of the invention.

In order to efficiently express the gene or gene fragment of the present invention, a promoter must be present in the expression vector. RNA polymerase normally binds to the promoter and initiates gene transcription. Promoters vary in potency, ie, the ability of the dc to promote transcription. Depending on the host cell system used, any of a variety of suitable promoters may be used. Suitable promoters include ubiquitin, the nos promoter, the ribulose bisphosphate carboxylase small subunit promoter, the chlorophyll A / B small subunit promoter, the cauliflower mosaic virus 35S promoter, and promoters isolated from plant genes. See C.E. Vallejos, et al., "Localization in the Tomato Genome of DNA Restriction Fragments Containing Sequences Homologous to the rRNA (45S), the major chlorophyll ab Binding Polypeptide and the Ribulose Bisphosphate Carboxylase Genes", Genetics 112: 93-105 (1986), which discloses small subunit materials. The nasal promoter and the 35S promoter of the cauliflower mosaic virus are well known in the art.

Once the disease resistance gene of the present invention is cloned into the expression system, it is ready to be transformed into the plant cell. Suitable plant tissues for transformation include leaf tissues, root tissues, meristems, and protoplasts.

Bacteria of the genus Agrobacterium can be used to transform plant cells. Suitable species of such bacteria include Agrobacterium tumefaciens and Agrobacterium rhizogens. Agrobacterium tumefaciens (e.g. strains LBA4404 or EHA105) is particularly useful for its well-known plant transformation capacity.

Another approach to transforming plant cells with genes involves introducing inactive or biologically active molecules into plant tissues and cells. This technique is disclosed in US Patent Nos. 4,945,050; 5,036.006; and 5,100,792 to Sanford et al. Generally, this procedure involves the injection of inactive or biologically active molecules into cells under conditions effective to penetrate the outer surface of the cell and allow incorporation into it. When inactive molecules are used, the vector may be introduced into the cell by coating the molecules with a vector containing the gene of interest. Alternatively, the target cell may be surrounded by a vector such that the vector is introduced into the cell following the molecule. Biologically active molecules (e.g., dried yeast cells, dried bacteria, or bacteriophage, each containing DNA that one wishes to insert) can also be inserted into the plant tissue.

The isolated gene fragment of the present invention can be used to confer disease resistance on a wide variety of plant cells, including gymnosperms, monocots and dicots. While the gene can be introduced into any plant cell belonging to these broad classes, it is particularly useful in cells of crops such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, beans, peas, chicory, etc. lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine,

187 851 apricot, strawberry, grapes, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybeans, tobacco, sorghum and sugarcane.

The expression system of the present invention can be used to transform essentially any crop plant under suitable conditions. The transformed cells can be regenerated into whole plants such that the gene confers disease resistance to entire transgenic plants. As set out above, the expression system may be modified such that the disease resistance gene is continuously or constitutively expressed.

Transformation.

The present system can be used in any plant that can be transformed and regenerated. Such transformation and regeneration methods are well known in the art. In addition to the references given above, see also G, Watson, B.D., and Chiang, C.C. Transformation of tobacco, tomato, potato, and Arabidopsis thaliana using a binary Ti vector system. Plant Physiol. 81: 301-305,186; Fry, J., Barnason, A., and Horsch, R.B. Transformation of Brassica napus with Agrobacterium tumefaciens based vectors. Pl. Cell Rep. 6: 321-325, 1987; Block, M.d. Genotype independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens. Theor. appl. genet. 76: 767-774,188; Deblock, M., Brouwer, D.D., and Terming, P. Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the Expression of the bar and neo genes in the transgenic plants. Plant Physiol. 91: 694-701,1989; Baribault, T.J., Skene, K.G.M., Cain, P.A., and Scott, N.S. Transgenic grapevines: regeneration of shoots expressing beta-glucuronidase. Pl. Cell Rep. 41: 1045-1049,1990; Hinchee, M.A.W., Newell, CA, ConnorWard, D.V., Armstrong, TA, Deaton, W.R., Sato, S.S., and Rozman, R.J. Transformation and regeneration of non-solanaceous crop plants. Stadler. Genet. Symp. 203212.203-212,1990; Barfield, D.G. and Pua, E.C. Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation. Pl. Cell Rep. 10: 308-314,1991; Cousins, Y.L., Lyon, B.R., and Llewellyn, D.J. Transformation of an Australian cotton cultivar: prospects for cotton improvement through genetic engineering. Aust. J.Plant Physiol. 18: 481-494. 1991; Chee, P.P. and Slightom, J.L. Transformation of Cucumber Tissues by Microprojectile Bombardment Identification of Plants Containing Functional and Nonfunctional Transferred Genes. GENE 118: 255-260,1992; Christou, P., Ford, T.L., and Kofron, M. The development of a variety-independent gene-transfer method for rice. Trends.Biotechnol. 10: 239-246,1992; D'Halluin, K., Bossut, M., Bonne, E., Mazur, B., Leemans, J., and Botterman, J. Transformation of sugarbeet (Beta vulgaris L.) and evaluation of herbicide resistance in transgenic plants. Bio / Technol. 10: 309-314. 1992; Dhir, S.K., Dhir, S., Savka, MA, Belanger, F., Kriz, A.L., Farrand, S.K., and Widholm, J.M. Regeneration of Transgenic Soybean (Glycine Max) Plants from Electroporated Protoplasts. PLANT PHYSIOL 99: 81-88,1992; Ha, S.B., Wu, F.S., and Thome, T. K. Transgenic turftype tall fescue (Festuca arundinacea Schreb.) Plants regenerated from protoplasts. Pl. Cell Rep. 11: 601-604,1992; Blechl, A.E. Genetic Transformation The New Tool for Wheat lmprovement 78th Annual Meeting Keynote Address. CEREAL FOOD WORLD 38: 846-847,1993; Casas, A.M., Kononowicz, A.K., Zehr, U.B., Tomes, D.T., Axtell, J.D., Butler, L.G, Bressan, R.A., and Hasegawa, P.M. Transgenic Sorghum Plants via Microprojectile Bombardment. PROC NAT ACAD SCI USA 90: 11212-11216,1993; Christou, P. Philosophy and Practice of Variety Independent Gene Transfer into Recalcitrant Crops. IN VITRO CELL DEV BIOL-PLANT 29P: 119-124,1993; Damiani, F., Nenz, E., Paolocci, F., and Arcioni, S. Introduction of Hygromycin Resistance in Lotus spp Through Agrobacterium Rhizogenes Transformation. TRANSGENIC RES 2: 330-335,1993; Davies, D.R., Hamilton, J., and Mullineaux, P. Transformation of Peas. Pl. Cell Rep. 12: 180-183,1993; Dong, J.Z. and Mchughen, A. Transgenic Flax Plants from Agrobacterium Mediated Transformation Incidence of Chimeric Regenerants and Inheritance of Transgenic Plants. PLANT SCI 91: 139-148,1993; Fitch, M.M.M., Manshardt, R.M., Gonsalves, D., and Slightom, J.L. Transgenic Papaya Plants from Agrobacterium Mediated Transformation of Somatic Embryos. Pl. Cell Rep. 12: 245-249,1993; Franklin, C.I. and Trieu, T.N. Transformation of the Forage Grass Caucasian Bluestem via Biolistic Bombardment Mediated DNA Transfer. PLANT PHYSIOL 102: 167,1993; Golovkin, M.V., Abraham, M., Morocz, S., Bottka, S., Feher, A., and Dudits, D. Production of Transgenic Maize Plants by Direct DNA Up187 851 take into Embryogenic Protoplasts. PLANT SCI 90: 41-52,1993; Guo, G.Q., Xu, Z.H., Wei, Z.M., and Chen, H.M. Transgenic Plants Obtained from Wheat Protoplasts Transformed by Peg Mediated Direct Gene Transfer. CHINA SCI BULL 38: 2072-2078. 1993; Asano, Y. and Ugaki, M. Transgenic plants of Agrostis alba obtained by electroporationmediated direct gene transfer into protoplasts, Pl Cell Rep. 13, 1994; Ayres, N.M. and Park, W.D. Genetic Transformation of Rice. CRIT REV PLANT SCI 13: 219-239,1994; Barcelo, P., Hagel, C. Becker, D., Martin, A., and Lorz, H. Transgenic Cereal (Tritordeum) Plants Obtained at High Efficiency by Microprojectile Bombardment of Inflorescence Tissue. PLANT J 5: 583-592,1994; Becker, D., Brettschneider, R., and Lorz, H. Fertile Transgenic Wheat from Microprojectile Bombardment of Scutellar Tissue. PLANT J 5: 299-307,1994; Biswas, GC.G, Iglesias, V.A., Datta, S.K., and Potrykus, 1. Transgenic Indica Rice (Oryza Sativa L) Plants Obtained by Direct Gene Transfer to Protoplasts. J BlOTECHNOL 32: 1-10,1994; Borkowska, M., Kleczkowski, K., Klos, B., Jakubiec, J., and Wielgat, B. Transformation of Diploid Potato with an Agrobacterium Tumefaciens Binary Vector System. 1. Methodological Approach. ACTA PHYSlOL PLANT 16: 225-230,1994; Brar, GS., Cohen, B.A., Vick, C.L., and Johnson, G.W. Recovery of Transgenic Peanut (Arachis Hypogaea L) Plants from Elite Cultivars Utilizing Accell (R) Technology. PLANT J 5: 745753,1994; Christou, P. Genetic Engineering of Crop Legumes and Cereals Current Status and Recent Advances. AGRO FOOD IND Hl TECH 5: 17-27, 1994; Chupeau, M.C., Pautot, V., and Chupeau, Y. 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Since their host plants may also be susceptible to pathogens outside the normal host range, these plants also have considerable utility in studying molecular, genetic and biological host-pathogen interactions. Moreover, the UDS phenotype of nim1 plants also makes them useful for fungicide studies. Nim1 mutants selected from individual hosts have considerable utility in screening fungicides using that host and host pathogens. The advantage lies in the mutant's UDS phenotype, which circumvents the problems encountered due to hosts being differentially sensitive to different pathogens or phenotypes, or even resistant to certain pathogens or pathotypes.

Pathogens of the invention include, but are not limited to, viruses or viroids, e.g., tobacco or cauliflower mosaic virus, ringspot virus, or necrosis virus,

Pelargonium leafroll, red spot virus, tomato bushy stunting virus, and similar viruses, fungi, e.g. Phythophthora parasitica and Peronospora tabacina; bacteria, e.g. Pseudomonas syringae and Pseudomonas tabaci, insects such as aphids, e.g. Myzus persicae; and Lepidoptera, e.g., Heliothus spp .; and nematodes, e.g., Meloidogyne incognita. The methods of the invention are effective against a variety of maize disease causing organisms including, but not limited to, downy mildews such as Scleropthora macrospora, Sclerophthora rayissiae, Sclerospora graminicola, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora sacchari, and Peronosclerospora sacchari; rusts such as Puccinia sorphi, Puccinia polysora and Physopella zeae; other fungi such as Cercospora zeae-maydis, Colletotrichum graminicola, Fusarium monoliforme, Gibberella zeae, Exserohilum turcicum, Kabatiellu zeae and Bipolaris maydis; and bacteria such as Erwinia stewartii.

SEQUENCE LIST DESCRIPTION.

SEQ ID NO: 1 - 9919 bp genomic sequence of Figure 14.

SEQ ID NO: 2 - 5655 bp genomic sequence of Figure 15.

SEQ ID NO: 3 - amino acid sequence of the wild NIM protein encoded by cds seq id no: 2.

SEQ ID NO: 4 - Amino acid sequence 33-155 from Rice-1 in Figure 19.

SEQ ID NO: 5 - Amino acid sequence sequence 215-328 from Rice-1 in Figure 19.

SEQ ID NO: 6 - Amino acid sequence 33-155 from Rice-2 in Figure 19.

SEQ ID NO: 7 - Amino acid sequence 208-288 from Rice-2 in Figure 19.

SEQ ID NO: 8 - Amino acid sequence 33-155 from Rice-3 in Figure 19.

SEQ ID NO: 9 - Amino acid sequence 208-288 from Rice-3 in Figure 19.

SEQ ID NO: 10 - Amino acid sequence 33-155 from Rice-4 in Figure 19.

SEQ ID NO: 11 - Amino acid sequence 215-271 from Rice-4 in Figure 19.

DEPOSITS.

The following vector molecules have been deposited with the American Type Culture Collection 12301 Parklawn Drive Rockville, MD 20852 U.S.A. on the dates given below:

Plasmid BAC-04 was deposited with the ATCC May 8, 1996 as ATCC 97543.

Plasmid P1-18 was deposited with ATCC on June 13, 1996 as ATCC 97606.

Kozmid D7 was deposited with the ATCC on September 25, 1996 as ATCC 97736.

EXAMPLES.

Example 1

Identification of NIM1 clones using map-based cloning. High-resolution genetic mapping and NIM1 physical mapping in Arabidopsis.

1. Plant material and the isolation of nim1 mutants.

Nim1 mutants were isolated from two Arabidopsis populations of the Ws-O ecotype as described by Delaney et al. (1995) PNAS 92, 6602-6606. One mutant population was an M2 library derived from ethylmethyl sulfonate (EMS) mutagenized seeds (purchased from Lehle, Round Rock, TX) and the other was a T-DNA population derived from seeds obtained from Ohio State University Arabidopsis Biological Resource Center (Columbus , OH).

The basis of the search for mutants with non-inducible resistance was the screening of mutagenized plant populations in which resistance to a virulent pathogen could not be induced with INA (2,6 dichloroisonicotinic acid; Metraux et al., 1991. In: Advances in Molecular Genetics of Plant-Mlcrobe Interactions, vol. 1, 432-439, Hennecke and Yenna, eds., Kessman et al., 1993 in: Mode of action of agrochemicals, Y. Honma, eds., Vernooij et al., 1995, Molec. Pl. Microbe Interaction 8, 228-234).

Plants from the mutant population were grown at high density in large trays in a commercial seeding mixture. When the plants were 2 weeks old, the trays were sprayed with 0.25 mg / ml INA. After four days, the plants were sprayed with a spore suspension of Peronospora parasitica, Em-Wa (EmWa) isolate, 5x10 ⁴ to 1x10 ⁶ spores / ml. This fungus is normally virulent on the ecotype

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Arabidopsis Ws-O, unless resistance is previously induced in these plants by INA or a similar compound.

After incubation in a high humidity environment, plants with visible disease symptoms were identified, generally 7 days after infection. These plants showed no resistance to the fungus despite the use of a resistance-inducing chemical, and were blown with potentially mutated plants with it (non-inducible resistance). Among 360,000 plants, 75 potential nim mutants were identified.

These potential mutants were then isolated from trays, placed under low humidity conditions, and allowed to set seeds. Plants derived from these seeds were screened identically for susceptibility to the fungus EmWa, again after pretreatment with INA. The progeny plants that showed symptoms were termed nim mutants. Six nim mutants were thus identified. One line (them) was isolated from the T-DNA population and five from the EMS population.

2. Determination of plant responses to INa and other chemical inducers of disease resistance.

i. Phenotypic analysis of them.

Salicylic acid (SA) and benzo (1,2,3) thia-diazole-7-carbothioic acid S-methyl ester are two chemical compounds that, like INA, induce a wide range of resistance known as Acquired Systemic Resistance (SAR) in wild type plants. Since INA did not induce resistance in plants therewith, disease resistance of these plants was also assessed after pretreatment with SA and BTH (as partially described in Delaney et al., 1995, PNAS 92, 6602-6606).

The plants were sprayed with 1, 5, or 15 mM SA or 0.25 mg / ml BTH and then assay inoculated with EmWa after 5 days (as described in Example 1 above). Both SA and BTH failed to protect the plants against fungal infection as evidenced by the presence of disease symptoms and fungal growth on these plants. Thus, plants did not respond to any SAR-inducing compounds, suggesting that the mutation occurred downstream of these compounds' entry into the resistance induction pathway.

They were also assessed for the response to infection with two incompatible isolates.

P parasitica, Wela and Noco (ie these fungal strains do not cause disease in wild Ws-O plants). The plants were sprayed with 5-10x10 ⁴ spores / ml Wela or Noco suspensions of conidia and incubated at high humidity for 7 days. Unlike wild-type plants, plants developed disease symptoms in response to both Wela and Noco infections. The symptoms were necrotic spots and stripes and some sporulation. After staining with lactophenol blue, it was easy to see fungal hyphae in the leaves of the plants with them. Thus, plants therein are susceptible to normally incompatible isolates of P. parasitica. This result shows that plants have a defect not only in chemically induced disease resistance but also in natural resistance to microorganisms that are usually non-pathogenic.

ii. Biochemical analysis of them.

SA, INA and BTH induce SAR and SAR gene expression, which includes genes associated with the pathogenesis of PR-1, PR-2 and PR-5 in Arabidopsis. Since these compounds did not induce resistance in them (as described in Example 1.2 above), the expression of the SAR genes in this lineage after SA, INA or BTH treatment was examined.

After the plants were treated with SA, INA or BTH, the plant tissue was harvested and the RNA accumulation from the PR-1, PR-2 and PR-5 genes was analyzed. For this, total RNA was isolated from the treated tissue and subjected to agarose gel electrophoresis. Three replica blots were prepared and each hybridized with a probe for one of the three genes as described in Delaney et al., PNAS 92, 6602-6606. Unlike wild-type plants, these chemicals did not induce RNA accumulation of any of these three SAR genes in plants with them, as shown in Figure 1. Together, these results indicate that the chemicals do not induce either SAR or expression of SAR genes in plants with them. .

Since the chemical compounds did not induce SAR or the expression of SAR genes in plants with them, it was interesting to see if infection with a pathogen could induce SAR gene expression in these plants as in wild-type plants. The Ws-O plants and them were sprayed with spo12

187,851 rye EmWa as described and tissues were collected for RNA analysis at several time points. Pathogen infection (EmWa) of wild-type plants induced expression of the PR-1 gene within 4 days after infection, as shown in Fig. 2. However, in plants with them, expression of the PR-1 gene was induced only 6 days after infection and levels at this time. it was lowered compared to the wild plant. Thus, in plants after infection with the pathogen, expression of the PR1 gene is delayed and decreased compared to the wild type.

Infection of wild-type plants by pathogens that induce a necrotic reaction leads to the accumulation of SA in the infected tissues. It has been shown that this endogenous SA is required for signaling in the SAR pathway, i.e. the degradation of endogenous SA leads to a reduction in disease resistance. This defines SA accumulation as a marker in the SAR pathway (Gaffney et al., 1993, Science 261, 754-756).

Plants were tested with them for their ability to accumulate SA after infection by the pathogen. Pseudomonas syringae tomato strain DC3000 carrying the avrRpt2 gene was injected into the leaves of 4-week-old plants therewith. Plants were harvested after 2 days for SA analysis as described by Delaney et al., 1995, PNAS 92, 6602-6606. This analysis showed that plants accumulated high levels of SA in infected leaves as shown in Figure 3. Uninfected leaves also accumulated SA, but not to the same level as infected leaves, similar to what was observed in wild-type Arabidopsis. This indicated that the nim mutation maps downstream of the SA marker in the signal transduction pathway. This was expected as lNA and BTH (inactive in plants by them) are known to stimulate a component of the SRA pathway downstream of SA (Vemoji et al., 1995, Molec. Pl. Microbe Interaction 8, 228-234; Friedrich et al., 1996 , The Plant Journal 8, on press; and Lawton et al., 1996, The Plant Journal 9, on press). Additionally, as described in Example 1.2, exogenously administered SA did not protect against EmWa infection.

3. Genetic analysis of them.

The plants were backcrossed to wild-type Ws-0 plants and the F1 progeny were tested for EmWa resistance after treatment with INA as described in Example 1.1. above. None of the F1 plants pretreated with INA showed disease symptoms while the control plants showed disease. So it was found that the mutation with them is recessive.

The F2 population from the Ws-0x them cross was also tested for disease resistance after pretreatment with lNA. Of this population, about 1/4 (32/130 plants) showed disease symptoms after treatment with EmWa of plants pre-sprayed with INA and 3/4 (98/130 plants) showed no disease at all. These results indicate that the nim mutation defines a single genetic locus and supports the Fl data which show the recessive nature of the mutation.

4. Identification of markers and genetic mapping of the NlM locus.

For conventional map-based NlM gene cloning, it was necessary to identify markers that were genetically closely linked to the mutation. This was done in 2 steps. First, plants were crossed with another Arabidopsis genotype, Landsberg erecta (Ler), and F2 plants from this cross were identified that had a phenotype with them (ie, plants that are homozygous for him / her at the NlM locus). Among them, plants were identified by molecular analysis that had the Lar genotype in a nearby DNA marker. Due to the identification criterion, these plants are recombinants between the marker and the NlM locus. The recombinant frequency determines the genetic distance between the marker and the NlM locus.

The second prerequisite for map-based cloning is to identify markers that are genetically very close to the NlM locus, i.e. markers that identify very few recombinants. If genetic markers that are very close are identified, they can be used to isolate genomic DNA clones that are close to the NlM locus. The NlM locus can then be cloned by chromosome walking, as long as it is not already present in the cloned DNA. Walking can start on either side of the gene. It is based on obtaining overlapping clones that are getting closer and closer to the gene of interest. When a single DNA marker is obtained from a walk started, for example, from the north end, and does not identify any recombinants between that marker and the gene of interest, it must be very close to the gene. However, if the marker identifies a recombinant (recombinants)

187 851 from the south end, the clone from which the marker was obtained had to cross the gene. By definition, the gene of interest had to be cloned. It must be located between this marker and the last marker at the north terminus that identifies the least number of recombinants at the north terminus. In the first step, a large number of recombinants are generated by genetic crossing. In a second step, recombinants close to the NIM1 gene are identified using molecular markers. Many markers have been described in the literature and there are several methods for obtaining additional markers. Our approach was based on a number of marker systems including SSLP and AFLP (see below).

i. Genetic crosswords.

To map the chromosome position of the NIM gene relative to the markers SSLP and AFLP, they were crossed with Ler to obtain the population for mapping. F2 plants were grown from this cross and the leaves collected for future DNA extractions. F2 plants were then tested for the nim1 phenotype as described in Example 1.1 above. F3 populations derived from individual F2 plants were also bred and tested for the nim1 phenotype. From the stored tissues of F2 and F3 plants with the nim1 phenotype, DNA was extracted by Ctab as described (Rogers and Bendich, 1986, Plant Molecular Biology Manual, A6, 1-10). This DNA was used for mapping the NIM gene as described above.

ii. Simple sequence length polymorphism markers.

Markers of simple sequence length polymorphism (SSLP) ATHGENEA and nga111 have been described (Bell and Ecker, 1994, Genomics 19, 137-144). The primers used to detect these SSLPs are given in Table 1.

Table 1

SSLP Primer Sequences

primer set primer sequence (5 'to 3') ATHGENEA (1) ACC ATG CAT AGC TTA AAC TTC TTG ACA TAA CCA CAA ATA GGG GTG C ATHGENEA (2) ACC ATG CAT AGC TTA AAC TTC TTG CCA AAT GTC AAA ATA CTC GTC nga 111 (1) CTC CAG TTG GAA GCT AAA GGG TGT TTT TTA GGA CAA ATG GCG nga 111 (2) CTC CAG TTG GAA GCT AAA G TGT TTT TTA GGA CAA ATG G

Genetic mapping of the NIM gene against the ATHGENEA marker.

Using ATHGENEA (1) primers for PCR amplification of Ler genomic DNA, a 205 base pair (bp) band was expected, while in Ws-0 genomic DNA a 211 bp genomic band (Bell and Ecker, 1994, Genomics 19, 137-144). The amplification products proved difficult to separate on conventional agarose gels. Therefore, two alternative methods have been developed for the separation and detection of these PCR fragments.

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In the first method, the ATHGENEA primer set (1) (Table 1) was used to amplify genomic DNA in the presence of 6-carboxyrodamine-labeled UTP (dUTPR110 obtained from ABI), which resulted in rhodamine-labeled PCR fragments. The pGr reactions were analyzed on a DNA Sequencer which detects DNA fragments with a single nucleotide resolution.

The specific reagents were 1 x PCR buffer, 2 mM MgCl 2, dNTPs 200 mM each, 2 mM dUTP-R110, ATHGENEA primers (1) 0.75 mM, 10 ng DNA and 0.75 units of Taq polymerase in a reaction volume of 20 ml. The amplification conditions were 3 minutes at 94 ° C followed by 35 cycles of 15 seconds at 94 ° C, 15 seconds at 55 ° C, and 30 seconds at 72 ° C. These samples were tested on an ABI 377 Sequencer capable of detecting fluorescently labeled DNA with a resolution of a single nucleotide (nt). This allowed genotyping of plant samples: a 205 nucleotide fragment was obtained from Ler DNA and a 211 nucleotide fragment from Ws-O DNA. Thus, fragments differing by 6 nucleotides in length could be easily distinguished, which allowed for easy genotyping of the samples as homozygous Ws-O, homozygous Ler and heterozygous Ws-O / Ler at the ATHGENEA locus.

A multiplexing scheme was used to increase the throughput of this system. Some DNA samples were amplified by PCR as described above with the ATHGENEA primer set (1), while other samples were analyzed with the ATHGENEA primer set (2) (given in Table 1), both in the presence of 6-carboxyrodamine labeled dUTP. The ATHGENEA primer set (2) was prepared based on the published ATHGENEA sequence (Simoens et al., 1988, Gene 67, 1-11). This primer set amplified a 139 bp DNA fragment from Ler DNA and a 145 bp band from Ws-O DNA. The amplification reaction conditions for the ATHGENEA primer set (2) were identical to those described for the ATHGENEA primer set (1) above.

Single reactions using the ATHGENEA primer set (1) and single reactions using the ATHGENEA primer set (2) were mixed together prior to electrophoresis on an ABI 377 Sequencer. This multiplex approach allowed genotyping 2 samples in a single Sequencer lane, one at positions 145/139 and one in positions 211/205 on the sequencer.

In the second method, PCR fragments were labeled with a primer labeled with the fluorescent dye FAM-6 (6-carboxyfluorescein) (Integrated DNA Technologies, Inc). The 5 'primers of the ATHGENEA primer sets (1) and (2) are identical in sequence (see Table 1). This primer was labeled with FAM-6 and used in a PCR amplification reaction with the following reagents (Perkin ELMER): 1 x XL buffer, 1 mM MgC12, dNTPs 200 mM each, 0.50 mM primers (5 'FAM-6 labeled primer), 10 ng genomic DNA and 0.5 units of XL polymerase in a reaction volume of 20 ml. The amplification conditions were 3 minutes at 94 ° C followed by 35 cycles of 15 seconds at 94 ° C, 15 seconds at 55 ° C, and 30 seconds at 72 ° C. Single reactions using the ATHGENEA primer set (1) and single reactions using the ATHGENEA primer set (2) were mixed together again prior to electrophoresis on the ABI 377 Sequencer. This multiplex approach allowed genotyping 2 samples in a single sequencer path, one at 145/139 nucleotides. and the other at position 211/205 nucleotides.

In all samples from plants with the nim phenotype, F2 and F3 plants were tested for their genotype for the ATHGENEA locus as described above. All samples that were heterozygous at this locus identified plants that were recombinant between the NIM1 locus and the ATHGENEA locus. In a population of 1144 F2 and F3 plants with the nim1 phenotype that were tested in this manner, 98 were heterozygous at the ATHGENEA locus, giving an estimate of the genetic distance between this SSLP locus and the NIM1 locus of 4.3 cM. It was therefore established that the NIM1 locus is on chromosome 1, near the ATHGENEA marker.

Genetic mapping of the NIM1 gene in relation to the nga111 marker.

Two sets of primers for the SSLP marker nga111 (described in Bell and Ecker, 1994, Genomics 19, 137-144) were used to amplify the genomic DNA of F2 and F3 plants with the nim1 phenotype and control Ws-O and Ler plants. The nga111 primer set (1) (described in Bell and Ecker, 1994, Genomics 19, 137-144 and listed in Table 1) was used under the following conditions: 1x PCR buffer, 2 mM MgC12, dNTPs each 200 mM, primers 0.75 mM . 10 ng of DNA and 0.75

187,851 units of Taq polymerase in a reaction volume of 20 ml. The nga 111 primer set (2) (given in Table 1), and the derivative of the ngal II primer set (l) were used under different conditions: 1 x PCR buffer, 1.5 mM MgC12, dNTPs 200 mM each, 1 mM primers, 10 ng DNA and 1 unit Taq polymerase in a reaction volume of 20 ml. Both reactions were amplified 1 minute at 94 ° C followed by 40 cycles of 15 seconds at 94 ° C, 15 seconds at 55 ° C and 30 seconds at 72 ° C.

The samples were analyzed on 3-5% agarose gels. The band obtained from the amplification of Ws-O DNA with both primer sets was 146 bp while Ler DNA amplification resulted in a 162 bp band. Plant samples that were heterozygous at the ngalll locus identified plants that were recombinant between this marker and the NIM locus. Among 1144 F2 and F3 plants phenotyped 239 were identified as heterozygous for the ngall1 marker, giving an estimate of the genetic distance between the SSLP marker and the 10.4 cM NIM locus. This confirmed that the NIMI locus is on chromosome 1. Since there were few plants with the phenotype of them that were heterozygous for both ATHGENEA and ngalll, it was determined that the NIMI locus lies between these two markers, ATHGENEA is located north of the NIMI gene and ngalll on south of the NIMI gene. This placed the NIMI gene approximately 10 cM north of ngalll, close to position 85 on chromosome 1 (Lister and Dean, 1993, Plant J. 4, 745-750; Bell and Ecker, Genomics 19, 137-144).

iii. Markers of the length polymorphism of amplified fragments.

For map-based NIMI gene cloning, it is necessary to identify the molecular markers that are getting closer to this gene. For this purpose, amplified fragment length polymorphism (AFLP) markers were generated by applying the restriction fragment selective amplification method described by Zabeau and Vos (1993, European patent application EP 0534858) and Vos et al. (1995, Nucleic Acid Research 23, 4407-4414) .

Outline of AFLP technology.

The use of AFLP technology in mapping is based on the selective amplification of a set of DNA bands in 2 genetically distinct samples. The finding that any bands obtained differ between the two genotypes identifies these bands as markers for that genotype. If the marker co-segregates at high frequency with the gene of interest (mutation) then the marker is near the genetic locus.

Selective amplification of a small set of DNA fragments in a complex DNA sample is achieved in 2 steps. First, DNA fragments are generated by digesting the DNA with restriction enzymes and then ligating the adapters to their ends. Second, primers composed of sequences complementary to the adapters plus a 3 'extension (typically 0-3 nucleotides) are used to amplify only those DNA fragments with ends that are complementary to these primers. If a single nucleotide extension is used, then each primer theoretically will fit about 1/4 fragments, and at both ends to 1/16. Thus, a limited set of DNA fragments is amplified with these primers. Further, by radioactively labeling one primer, an even smaller subset of the visible fringes can be obtained.

AFlP analysis

For AFLP analysis of DNA samples, 50 ng was digested with the appropriate enzymes (generally EcoRI and Msel, see below) and adapters (given in Table 2 below) were attached to the restriction fragments (generally EcoRI and Msel). The sequences of the YAC, P1 and BAC primers and clones are described in detail below. The templates were used for the amplification reaction (approximately 0.5 ng DNa per reaction) using primers that were complementary to the adapters with short 3 'extensions (2 or 3 nucleotides; primer sequences are given below). Since one of the primers is radiolabeled (generally the EcoRI primer), only a subset of the amplified fragments is visible on autoradiographic analysis of the gel used to separate the bands.

The following conditions for the amplification of the cloned DNA were used (YAC, PI, cosmid): 36 cycles 30 sec. at 94 ° C (denaturation), 30 sec. hybridization and extension 1 min. at 72 ° C. The annealing temperature for the first cycle was 65 ° C and was decreased by 0.7 ° C in each cycle for the next 12 cycles and then held at 56 ° C. For genomic

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DNA from Arabidopsis plants was amplified in two steps: in the first (preamplification), DNA was amplified with primers that had a single nucleotide extension (none of the primers were labeled). The reaction conditions for this amplification reaction were: 20 cycles of 30 sec. denaturation (94 ° C), 1 min. hybridization (56 ° C) and 1 min. elongation (72 ° C). In the second step, the first amplification reaction was diluted 10 times and reamplified for 36 cycles with primers containing full-length extensions (using one labeled primer) under the following conditions: 30 sec 94 ° C (denaturation), 30 sec. hybridization and 1 min. elongation (72 ° C). The annealing temperature for the first cycle was 65 ° C and was decreased by 0.7 ° C in each cycle for the next 12 cycles and then held at 56 ° C. The end products of the reaction were separated on a polyacrylamide gel and the gel exposed to a plate allowing the visualization of radiolabeled PCR bands. When this procedure was applied to DNA from 2 genotypes simultaneously, AFLP bands were identified that were diagnostic for one genotype or the other. Such bands are called AFLP messenger bands, or AFLP markers. Table 2 shows the adapters used for AFLP analysis.

Table 2

enzyme adapter EcoRl 5'-CTC'GTAGACTGCGTACC-3 ' 3'-CATCTGACGCATGGTTAA-5 ' Hindlll 5'-CTCGTAGATGCGTACC-3 ' 3'-CATCTGACGCATGGTCGA-5 ' Pstil 5'-CTCGTAGACTGCGTACATGCA-3 ' 3 '-CATCTGACGCATGT-5' Msel 5'-GACGATGAGTCCTGAG-3 ' 3 '-TACTCAGGACTCAT-5'

Generation of AFLP markers and detailed mapping of the NlM1 locus.

A population of recombinant inbred lines derived from a cross between the Arabidopsis Landsberg erecta (Ler) and Columbia (Col) ecotypes (Lister and Dean, 1993, Plant J. 4, 745-750) was used to search for AFLP markers. The following primers were used for the AFLP test:

- EcoRl primers: 5'-GACTGCGTACCAATTCWN-3 '

- Msel primers: 5'-G ATGAGTCCTGAGTAAXWN-3 '"N" in primers means that this part is variable (A, C, G or T), "W" means A or T, and X means C. All 8 possible primers were applied to both the EcoRI and Msel primers. This resulted in a total of 64 (8x8) primer combinations (PC) that were used to amplify DNA from the recombinant inbred line and the parental Ler and Col genotypes as described above. The amplification reactions were electrophoresed on a denaturing polyacrylamide gel to separate the AFLP fragments by size, and the gel was exposed to film. The presence of bands in only one genotype was checked on the film, i.e. AFLP markers were searched.

AFLP markers, i.e. DNA fragments that are polymorphic between both parents of recombinant inbred lines, were used to construct a genetic map of the recombinant inbred population. Example 1.5i below describes the mapping of the NlM1 gene on Arabidopsis chromosome 1, approximately at position 85. Those AFLP markers that were mapped (using a recombinant inbred line) between positions 81 and 88 on the Arabidopsis chromosome were selected to analyze recombinant plants for the presence of said

187,851 AFLP markers thus for a more accurate mapping of the NIM1 gene. Seven informational AFLP markers have been identified in this region; were polymorphic between both parents of the nim1 x Ler cross. The six AFLP markers were Ler specific, i.e. these markers were absent in Ws (and in Col). One AFLP marker was Ws specific, i.e. a Col ratio specific AFLP marker (not present in Ler) was also present in Ws. These AFLP markers are: L81.1, L81.2, W83.1, L84., L85, L87 and L88 (marker L is specific for the Ler ecotype and marker W is specific for both the Col and Ws ecotypes; the number indicates the position on the map ). These AFLP markers were used to analyze recombinant plants from the nim1 x Ler cross (see below). In addition, the marker AFLP C86 (a Col-specific recombinant inbred marker) was used to isolate DNA clones (see below). Table 3 lists the primer sequences that were used to obtain these AFLP markers.

Table 3 lists the primer combinations derived from the recombinant inbred population.

"EcoRI" refers to the 5'-GACTGCGTACCAATTC-3 'sequence and "Msel" refers to the 5'-GATGAGTCCTGAGTAA-3' sequence.

Table 3

AFLP marker corresponding combinations of primers L81.1 EcoRI-CA Msel-CCG L81.2 EcoRI-AA Msel-CAA W83.1 EcoRI-CA Msel-CTC L84 EcoRI-AAT Msel-CAA L85 EcoRI-CA Msel-CCT L87 EcoRI-CA Msel-CTT L88 EcoRI-AG Msel-CTA C86 EcoRI-AG Msel-CCT

A detailed genetic map of the region was constructed using the AFLP markers described above by recombinant typing. In total, 337 recombinant plants were available from 1144 F2 nim1 plants. These recombinants were first tested with the north flanking markers AFLP L81.2 and ATHGENEA and the south flanking markers L88 and nga111. Forty-eight plants were homozygous for nim1 / nim1 and heterozygous for ATHGENEA and L81.2, 21 plants were homozygous for nim1 / nim1 and heterozygous for nga111 and L88. These recombinant plants were further analyzed with 9 AFLP markers in the NIM region, including 4 AFLP markers derived from the recombinant inbred mapping population (W83.1, L84, L85 and L87) and 5 AFLP markers derived from YAC clone analysis (W83.2 / W84.1, W84.2, W85.1, W86.1 and L86, see below).

The genetic map of NIM1 based on this analysis is shown in Figure 4. As can be seen, 27 recombinants were found between marker W84.2 and NIM1 and 14 recombinants between W85.1 and NIM1. The L85 marker is closely linked to NIM1, but this marker could not be mapped to the YAC, BAc or P1 clones (see below) and was therefore not useful for identifying the NIM1 gene.

5. Physical mapping of the NIM1 area.

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i. Isolation of YAC clones using AFLP markers closely linked to NIM1.

The CIC library, the Columbia ecotype YAC Arabidopsis library (Bouchez et al., 1995, 6th Int. Conf. On Arabidopsis Research, Madison, WI) was tested for YAC clones in the NIM region. The library is approximately 2.5 equivalents of the nuclear genome and has an average insert size of 450 kb. The YAC library was tested with two AFLP markers: W83.1 and C86. W83.1 is the most closely linked recombinant inbred AFLP marker north of NIMI, and C86 is a Col specific recombinant inbred AFLP marker (absent in Ler and Ws). C86 mapped south of the NIMI gene on the recombinant inbred population map. This Col AFLP marker was used in place of the closely linked Ler AFLP markers (Figure 4) because the latter AFLP markers only detected the Landsberg erecta ecotype and therefore cannot be used to screen the Columbia YAC library.

The YAC library was tested in two steps. First, YAC clone cells from each plate from 12 96-well microliter plates were pooled (plate pool) and used for DNA isolation as described by Ross et al. (1991, Nu ^ eic Acids Res., 19, 6053). Pools were tested with both AFLP markers. Then, samples of each row (8 clone pool) and each column (12 clone pool) were tested from each positive plate pool with the AFLP marker for which the plate pool was positive. In this way, individual positive YAC clones could be identified. The searches resulted in a total of 4 YAC clones: YAC 12F04 and YAC 12H07 were isolated using the North marker AFLP W83.1 and YAC 10G07 and YAC 7E03 using the southern marker C86 (CIC numbering is used in the nomenclature of the YAC clones). The YACi were fingerprinted with AFLP which gave YAC-specific AFLP fragments. YAC fingerprints were compared and used to estimate overlaps between YACs (see also Tables 5 and 6). Based on the AFLP fingerprints, clone 7E03 is essentially covered by clone 10G07 (see also Table 5) and clone 12H07 is essentially covered by clone 12F04 (see also Table 6).

ii. Generation of AFLP markers from YAC clones.

Since the AFLP markers described above were genetically quite distant from the NIMI gene (see Figure 3), additional AFLP markers were developed to find markers closer to the NIM gene.

The search for additional YAC-derived AFLP markers was performed on the following DNA samples: DNA of isolated YAC clones (4 YACs identified as described above), YAC-free yeast strain, and three Arabidopsis Col, Ler and Ws ecotypes. In this way, fragments specific for YAC clones (not present in the yeast strain and present in Col) could be tested for polymorphism in Ler and Ws (parents of the recombinant plants defined in Example 1.5 below). All identified polymorphic fragments would therefore be additional AFLP markers. The first study on AFLP used the enzyme combination (EC) EcoRI / Msel. Two YAC clones (10G07 and 7E03 (detected by the AFLP marker C86, see below), a yeast strain without YAC and three Arabidopsis Col, Ler and Ws ecotypes were tested. Primer combinations with selective extensions can be divided into three groups and are shown in Table 4. A total of 256 (64 + 96 + 96) primer combinations were tested.

Table 4 below lists the primer combinations used for the AFLP study of the two YAC clones 10G07 and 7E03, the yeast strain without YAC and the three Arabidopsis Col, Ler and Ws ecotypes. Three groups of primer combinations were used. "N" in the primers means that part is variable (A, C, G or T), "S" means C or G, "W" means A or T and "Y" means C or T.

Table 4 EcoRI primers:

5'-GACTGCGTACCAATTCGW-3 '5'-GACTGCGTACCAATTCTS-3' Msel primers:

5'-GATGAGTCCTGAGTAAAAS-3 '5'-GATGAGTCCTGAGTAAASA-3' 5 '-GATGAGTC CTGAGTAAATN-3'

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c.d. table 4

5'-GATGAGTCCTGAGTAACAN-3 '

5'-GATGAGTCCTGAGTAACTN-3 'EcoRI primers:

5'-GACTGCGTACCAATTCAN-3 '

5'-GACTGCGTACCAATTCCW-3 '' -GACTGCGTACC A ATTC C W-3 'Msel primers:

5'-GATGAGTCCTGAGTAAAAS-3 '

5'-GATGAGTCCTGAGTAAASA-3 '

5'-GATGAGTCCTGAGTAAGAY-3 '

5'-GATGAGTCCTGAGTAAGTW-3 '

5'-GATGAGTCCTGAGTAATCG-3 '

5'-GATGAGTCCTGAGTAATCT-3 '

5'-GATGAGTCCTGAGTAATGW-3 'EcoRI primers:

'-GACTGCGTACC AATTCG W-3' 5'-GACTGCGTACCAATTCTN-3 'Msel primers:

5'-GATGAGTCCTGAGTAAGAW-3 '

5'-GATGAGTCCTGAGTAAGCW-3 '

5'-GATGAGTCCTGAGTAAGTW-3 '

5'-GATGAGTCCTGAGTAATAN-3 '

5'-GATGAGTCCTGAGTAATCW-3 '

5'-GATGAGTCCTGAGTAATGW-3 '

5'-GATGAGTCCTGAGTAATTS-3 '

A total of 83 Col specific fragments were generated, 62 of which were common to both YAC clones. Three fragments were AFLP markers polymorphic between Ws and Ler, two of which were AFLP markers Ws (Col fragment also present in Ws and not present in Ler) and one was a Ler AFLP marker (Col fragment also present in Ler and absent in Ws). The results are shown in Table 5 below.

Table 5 shows the number of common and unique AFLP fragments detected in YACs 10G07 and 7E03 and the number of informative AFLP markers among these fragments in the Ws and Ler genotypes.

Table 5

AFLP fragments in YAC clones AFLP marker 10G07 7E03 Ws Ler common 62 62 2 1 unique 21 0 0 0

This AFLP analysis thus gave 3 new AFLP markers (see figure 4 and below). Their positions relative to each other and relative to the AFLP markers derived from the recombinant inbred line were determined by analysis of recombinants with these AFLP markers.

A second search for AFLP markers is shown by marking all four YAC clones found (see below) using the Pst / Msel enzyme combination.

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The following primers were used: Pstl primers:

5'-GACTGCGTACATGCAGAN-3 '

5'-GACTGCGTACATGCAGCW-3 '

5'-GACTGCGTACATGCAGGW-3 '

5'-GACTGCGTACATGCAGTN-3 'Msel primers:

5'-GATGAGTCCTGAGTAAAN-3 '

5'-GATGAGTCCTGAGTAACW-3 '' -GATGAGTCCTGAGTAAGW-3 '

5'-GATGAGTCCTGAGTAATN-3 '"N" in primers means that part is variable (A, C, G or T), "W" means A or T. A total of 144 (12x12) primer combinations were tested for all four isolated YAC clones: 12F04, 14H07, 10G07 and 7E03; yeast strain without YAC; and the three ecotypes of Arabidopsis Col, Ler and Ws. A total of 219 AFLP fragments were generated, of which 144 were present in the YAC clones 12F04 and 12H07 (72 were unique to clone 12F04 and 72 were shared by both YACs), of which 75 were present in the YAC clones 10G07 and 7E03 _ (33 were unique for clone 10G07 and 42 were common to 2 YACs). The three fragments derived from the first set of YAC clones were polymorphic (Ws AFLP markers). The results are shown in Table 6 below.

Table 6 shows the number of common and unique AFLP fragments detected in the YACs and the number of AFLP informational markers among these fragments in the Ws and Ler genotypes.

Table 6

number of AFLP fragments in YAC clones AFLP markers 12F04 12H07 10G07 7E03 Ws Ler common 72 72 0 0 1 0 unique 72 0 0 0 2 0 common 0 0 42 42 0 0 unique 0 0 33 0 0 0

The results show that the YAC 12H07 clone is part of the larger YAC 12F04 clone and that the YAC 7E03 clone is part of the larger YAC 10G07 clone. These data demonstrate that the larger YAC clones 12F04 and 10G07 do not overlap. These data do not allow the NlM gene to be placed on any of these YAC clones. The entire search involving 400 combinations of primers producing 302 AFLP fragments in the N1M region yielded 5 useful AFLP markers, 4 of which were Ws specific and one Ler specific. These 5 additional AFLP markers were mapped by analysis of recombinant plants (see figure 4 and below) and were named W84.1 (also called W83.3), W84.2, W85.1, W86.1 and L86.

Table 7 lists the primer sequences used to generate these AFLP markers. These 5 additional AFLP markers led to 12 total AFLP markers in the region from L81.1 to L88 (see figure 4 and below).

Table 7 shows the combinations of AFLP markers derived from YAC clones.

"EcoRl-" refers to the 5'-GACTGCGTACCAATTC-3 'sequence, "Msel-" refers to the 5'-GATGAGTCCTGAGTAA-3' sequence and "Pstl-" refers to the 5'-GACTGCGTACATGCAG-3 'sequence

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Table Ί

AFLP marker primer combination with selective extension W84.1 Pstl-AT Msel-TT W84.2 Pstl-AA Msel-TT W85.1 EcoRI-CT Msel-GTA W86.1 EcoRI-GT Msel-CTT L86 EcoRI-GT Msel-CTT

This information was used to construct a physical map of the area as shown in Figure 5 with the approximate positions of the YAC clones relative to the genetic map. The map showed that the region containing the NIM1 locus between markers W83.1 and W85.1 is partially covered by 3 YAC clones: 12F04 and 10G07 / 7E03.

iii. Construction of a P1 / BAC contig containing the NIM1 gene.

The previous sections described how AFLP markers linked to the NIM1 region were isolated and how the YACi corresponding to these markers were identified and mapped. The results obtained during the localization of the NIM1 gene to the chromosome fragment did not allow the determination of the specific DNA segment containing the NIM.1 gene: the flanking AFLP markers were mapped to different YAC clones that did not overlap. Thus, it was not possible to determine the exact physical position of the NIM1 gene; it could be located on any of the YACs or in a gap between YACs. An alternative approach was chosen to close the physical gap between the flanking markers: P1 and BAC libraries were used to close the gap between the flanking AFLP markers

The libraries used to close the library were the Columbia ecotype P1 library described by Liu et al. (The Plant J. 7, 351-358, 1995) and the Columbia ecotype YAC library described by Choi et al. (Http / genome-www.stanford .edu / Arabidopsis / ww / vol2 / choi.html). The P1 library consists of approximately 10,000 clones with an average insert size of 80 kb and the BAC library consists of approximately 4,000 clones with an average insert size of 100 kb. Theoretically, these libraries represent approximately 10 nuclear genome equivalents (assuming a haploid Arabidopsis genome size of 120 Mb).

iv. Identification of P1 clones corresponding to flanking markers.

The flanking markers Ws84.2 and Ws85.1 were used to screen the P1 clone pool using a similar strategy to that previously described for testing the YaC library (see example 1.5i). P1 clones having marker fragments were selected and "plasmid" DNA was isolated. The DNA of the various P1 clones was fingerprinted using EC EcoRI / Msel and HindID / Msel and primers without selective nucleotides. A physical map, i.e. a map giving sizes and clone tabs, was constructed by comparing the AFLP fingerprints. The number of aFLp fragments that are unique and the number of fragments that are common between clones indicate the extent of the overlaps. The map is shown in Figure 6. The AFLP fingerprints showed that two sets of non-overlapping P1 contigs were constructed, each containing one of the flanking markers: P1-1 and P1-2 containing the Ws84.2 marker; P1-3 and P1-4 containing marker W85.1. Thus, the gap between the flanking markers was not closed (figure 6).

The position of the P1 contigs in relation to the YAC contig was determined by AFLP fingerprinting of the YACs and P1 clones using the YAC-specific PC series described above. Clones P1 P1-1 and P1-2 appeared to completely overlap YAC CIC12F04 but only partially overlap YAC CIC12H07 / 12F04 (figure 6). Clones P1 P1-3 and P1-4 completely overlapped both YACi CIC7E03 and CIC10G07 and the AFLP marker W86.1 like W85.1 appeared to map in this P1 contig (figure 6).

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The marker L85 was then used to identify the corresponding P1 and BAC clones. L85 is a marker specific for the Landsberg ecotype, so colony hybridization of radioactively labeled L85 DNA was applied to the P1 and BAC filters. Not a single clone of P1 nor was found. BAC hybridizing to L85. This supported our earlier finding that the L85 sequence was not present in the Columbia Arabidopsis ecotype and is therefore the most likely explanation for why no suitable clones were found.

v. Extension of the P1 contigs surrounding NlM1.

Various approaches have been used to extend the surrounding P1 contigs.

YAC AFLP fragments specific for the south end of YAC ClC12F04 (unique to ClC12F04, not present in ClC12H07) were used to identify P1 clones by AFLP screening of the library pool.

1. YAC AFLP fragments from YAC 10G07 and overlapping P1-4 were used to identify P1 clones by AFLP screening of the P1 library pool.

2. EcoRl restriction fragments from P1 P1-6 clones (resulting from the AFLP-based P1 library screening of step 1) were used as probes for hybridization to the BAC library filters.

Different P1 and BAC clones resulted from this search and all were analyzed by AFLP fingerprinting with EC EcoRl / Msel and HindIII / Msel using primers without selective nucleotides. A new map was constructed which is shown in Fig. 7 Table 8 shows the different AFLP PCs having fragments AFLPs mapped to flanking YACs and used to screen the P1 library for appropriate P1 clones.

Table 8 shows the different AFLP PCs used to screen the P1 library. The top half of the table shows the PCs specific to the North YAC and the bottom half shows the PCs specific to the South YAC. Also shown are YAC and P1 clones in which AFLP fragments were detected

Table 8

AFLP PC Pstl-AA Msel-TT Pstl-AT Msel-GT Pstl-CA Msel-AA Pstl-AC Msel-TG Pstl-AG Msel-TG Pstl-CT Msel-GT ClC YAC 12F04 and 12H07 12F04-specific 12F04-specific 12F04-specific 12F04-specific 12F04-specific P1 clones P1-1.P1-2 P-1.P1-2 P1-6 P1-7 P1-7 P1-7 Comments Ws84 marker. 2 EcoRl-CT Msel-GTA 10G07 and 7E03 P1-3, P1-4 Ws85.1 marker EcoRl-GT Msel-CTT 10G07 and 7E03 P1-4 Vs86.1 marker EcoRl-AA Msel-GT 10G07 and 7E03 P1-4, P1-9 EcoRl-AT Msel-GA 10G07 and 7E03 P1-4P1-9 EcoRI-GG Msel-CT 10G07 and 7E03 P1-4, P1-9

The obtained P1 / BAC contig was approximately 250 kb spanning the south end of the YAC ClC 12F04 (not extending beyond this YAC) and containing the marker W84.2. A P1 contig of approximately 150 kb containing the markers W85.1 and W86.1 was obtained; this contig is completely contained in YAC ClC7E03.

The construction of the P1 / BAC contig covering the AFLP markers of the NlM1 gene on recombinants from the markers from the south-north end of the P1 / BAC contig (WL84.4 and WL84.5, see below and Table 11) showed that the previous steps of "walking" were not successful in the construction contig containing the NlM1 gene (see next section). Thus, the existing PlBAC north contig was extended south to "pass" through the NlM1 gene, which would allow the definition and isolation of a specific DNA segment containing the NlM1 gene. A hybridization-based approach was used in which clones on P1 or BAC located at the south end of the northern P1 / BAC contig were used to identify clones closer to NIM1 (southbound). New clones resulting from the cutting steps were mapped against the existing contigs using AFLP fingerprints from EC EcoRI / Msel and HindIII / Msel as described above. A total of 5 consecutive traversing steps proved necessary for the NIM1 gene "transition". Table 9 shows the clones obtained in the different walking steps.

Table 9 gives an overview of the different walking steps showing the hybridization probe used to screen the P1 and BAC libraries and selected clones hybridizing to the probes and extending southward.

Table 9

Probe New clones reaching south Level 1: P1-7 BAC-02 Stage 2: BAC-02 P1-16, BAC-03 Stage 3: BAC-03 P1-17, P1-18 Stage 4: P1-18 P1-21, P1-20, BAC-04 Stage 5: BAC-04 P1-22, P1-23, P1-24, BAC-06, BAC-05

The physical map of the various clones resulting from this walk is shown in Figure 8. In total, a total distance of 600 kb was covered starting from the starting walking point, marker W84.2. The southern end of the contig shown in Figure 8 appeared to contain the NIM gene (see next section). The contig extends over 300 kb south of the YAC CIC12F04 and does not appear to overlap with the YACi CIC10G07 and CIC7E03, indicating that the NIM1 gene is in the gap between the flanking YAC contigs and the gap is at least 300 kb.

vi. Construction of an integrated genetic and physical map of the NIM1 region.

The previous sections described how the AFLP markers linked to the NIM1 region were isolated, how the YACi corresponding to the flanking markers were identified, and how the P1 / BAC contig extending about 550 kb south of the nearest flanking AFLP marker W84.2 was constructed. This section describes the generation of new AFLP markers from the PlBAC contig, the physical mapping of these markers on this contig, and the genetic mapping of these markers with available recombinants.

1. Generation of new AFLP markers from the P1 / BAC contig.

As described in the previous section, P1 and BAC clones from the contig extension were characterized by AFLP fingerprinting using EC EcoRI / Msel and HindIII / Msel. This quite accurately determined the degree of overlap between the different P1 and BAC clones and additionally generated a number of AFLP fragments specific to these clones. AFLP primers without selective nucleotides are used in the fingerprinting of purified plasmid DNAs of P1 or BAC clones. Selective nucleotides are, however, necessary to use these P1 or BAC specific AFLP fragments for detection in Arabidopsis. By determining the end sequences of the amplified restriction fragments, AFLP primers having appropriate selective bases for the amplification of a P1 or BAC specific AFLP fragment in Arabidopsis can be designed. All AFLP fragments are derived from the Columbia (Col) ecotype, and thus it should also be ascertained whether the Columbia AFLP markers are informative for NIM1 recombinants that are derived from a cross between Landsberg erecta (Ler) ecotype and the nim1 mutant of the Wassilewskija ecotype (Ws-nim). In principle, there are four types of AFLP fragments, two of which are useful markers as listed in Table 10 below:

Table 10 gives an overview of the types of AFLP markers found. (+) and (-) indicate the presence or absence of an AFLP fragment

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Table 11

Col Ler In-him marker type + + + not informative + + - marker Ler + - + Ws marker + - - not informative

In general, fingerprinting of P1 and BAC clones generated 30 to 40 AFLP EcoRI / Msel fragments and 60 to 80 AFLP HindIII / Msel fragments for each individual clone. These final sequences of the individual fragments were determined using standard sequencing methods. The specific AFLP primer sets were then tested with selective 3 nucelotide extensions for both the EcoRI or HindIII primers and the Msel primer on the following DNA set:

1. The Pl / BAC clone from which the AFFLP marker was derived

2a. Yeast

2b. YAC clone CIC12F04 (only for AFLO fragments from P1-7)

2c. YACCIC10G07 clone

3a. Col from which the P1 and BAC libraries are derived

3b. Ler, parent of the 1st recombinants of him

3c. Ws-nim, parent of the 2nd recombinants in him

6 clones were selected for sequence analysis of their AFLP EcoRI / Msel and HindIII / -Msel fragments: BAC-01 / P1-7, P1-P17 / P1-18, BAC-04 / BAC-06. All AFLP fragments from clone P1-P7 were detected in YAC CIC12F04, indicating that this clone is completely contained in this YAC. None of the P1 / BAC specific AFLP fragments were detected in the YAC CIC 10G07 clone, indicating that the P1 / BAC contig does not bridge the gap between the flanking YAC contigs. The AFLP markers selected for recombinant analysis are shown in Table 11.

Table 11 gives an overview of selected AFLP markers from PC AFLP specific for different P1 and BAC clones. The "WL" marker is a marker derived from the same PC and showing two AFLP markers, Ws and Ler marker, which were found to be fully repulsion-linked when analyzed for NIM recombinants.

Table 11

Origin Marker name Combination of AFLP primers P1-7 WL84.4 EcoRI-AGC Msel-ACT P1-7 WL84.5 Hindlll-CTC Msel-TTC P1-17 / P1-18 Ler84.6a Hindlll-CGT Msel-ATT P1-17 / P1-18 Ler84.6b Hindffl-ATT Msel-CAT P1-18 Ler84.6c Hindlll-TCT Msel-TAT P1-18 Ler84.7 EcoRI-AAA Msel-AGA BAC-04/06 Ler84.8 EcoRI-TTC Msel-AGT BAC-06 Ler84.9a EcoRI-AAA Msel-TGT BAC-06 Ler84.9b EcoRI-ATC Msel-TCC BAC-06 Ler84.9c EcoRI-ATG Msel-GTA

2. Physical mapping of new AFLP markers

The AFLP markers described above were physically mapped by detecting their presence in different P1 and BAC clones. The results are shown in Figures 9-11.

3. Genetic mapping of new AFLP markers.

AFLP markers were analyzed in a selected set of recombinants. The results obtained are summarized in Tables 12a, 12b and 12c.

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The AFLP markers Ler84.8, Ler84.9a, Ler84.9b, and Ler84.9c seemed to map to the south of NlM1. Recombinants were found that were phenotypically nim1 (homozygous, genotype Ws-nim1 / Ws-nim1) and heterozygous for these AFLP markers (Ler-specific AFLP marker detected, genotype is Ws-nim1 / Ler). The AFLP marker Ler84.8 appeared to be the closest to NlM1: only one recombinant was found as heterozygous Wsniml / Ler and homozygous Ws-nim1 / Ws-nim1. The AFLP markers Ler84.7 and Ler84.6 appeared to completely co-segregate with NlM1: all recombinants had identical genotype of NlM1 and AFLP markers. NA north of NlM1, marker L84.6b appeared closest to NlM1: three recombinant plants with the nim1 phenotype: C-074, D-169 and E-103 (Table 12c) appeared to be Ws-niml / Ler heterozygous in this marker. Using the cosmid contig generated from P1-18; BAC-04 and BAC-05, the AFLP markers Ler84.6b and Ler84.8 were mapped to P1-18 and bAC-04, respectively, and were found to have a physical distance of approximately 110 kb. This defines that the nim1 locus is located on a stretch of DNA that is estimated to be 110 kb in length. From this analysis, the NIM1 gene was found to be contained in the BAC-04 or P1-18 clone. Clones BAC-04 and P1-18 have been deposited with ATCC and have been assigned ATCC numbers 97543 and ATCC 97606, respectively.

vii. Detailed genetic and physical mapping of the NlM1 gene.

The previous section describes how a stretch of DNA containing the NlM gene was determined by physically mapping the flanking markers (Ler84.6b and Ler84.8) on the P1 / BAC contig. The flanking markers seemed to map to the two overlapping clones P1-18 and BAC-04. This section describes how additional AFLP markers specific for BAC-04 and P1-18 were generated to increase the resolution of the genetic and physical map in the region containing the NlM1 gene.

viii. Generation of additional AFLP markers from the cosmid representation.

Four ECs were selected to generate further AFLP markers for detailed NlM1 mapping: Pstl / Msel, Xbal / Msel, BstYl / Msel and Taql / Msel. AFLP PstI / Msel and Xbal / Msel fragments were generated on clone P1-18 and BAC-04 and the selective sequences needed for detection in Arabidopsis were determined. Similarly, AFLP fragments and sequences selective for BstYl / Msel and Taql / Msel were established; however, in this case, the procedure was performed using cosmid DNA: A11, C7, E1 and E8 for BstYl / Msel (total NlM1 area) and D7, E8 and E6 for Taql / Msel (southern part of NlM1 region). The informational AFLP markers selected for further genetic and physical mapping are shown in Table 13. Additional adapters used in this work are shown in Table 14.

Table 13 shows the AFLP markers used for the detailed genetic and physical mapping of NlM1. "BstYl (T)" indicates that the restriction site and the corresponding primer were either AGATCT or GGATCT.

Table 13

Marker EC / PC Ler84.Yl BstYl (T) -GCT Msel-AAC Ws84.Y2 BstYl (T) -TCT Msel-GCA Ler84.Y3 BstYl (T) -AAG Msel-TAT Ler84.Y4 BstYl (T) -TT Msel-AGA Ws84.Tl Taql-TAC Msel-GGA Ler84.T2 Taql-TTG Msel-GGA

Table 14 shows some additional adapters used to identify new AFLP markers.

Table 14

BstYl: 5'-CTCGTAGACTGCGTACC-3 '3'-CATCTGACGCATGGCTAG-5'

Taql: 5'-CTCGTAGACTGCGTACC-3 '3'-CATCTGACGCATGGGC-5' ix. Physical mapping of new AFLP markers to the cosmid contig.

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The above-indicated markers were physically mapped to the cosmid representation by determining their presence in the various cosmid clones (Figure 11).

1. Genetic mapping of new AFLP markers.

New AFLP markers were genetically mapped by AFLP analysis of the nearest northern and southern recombinants. The closest northern (recombinant D169) and southern (recombinant C105) recombination points (see Table 15) are mapped. AFLP analysis showed that recombinant D169 had recombination south of the marker L84.Y1 but north of the marker W84.Y2. The recombination point in recombinant C105 mapped between markers L84.T2 and L84.8. The use of the available set of recombinants allowed for further determination of the chromosome segment containing NIM1; the distance between the flanking points appeared to be 60-90 kb (Figure 12).

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2. Construction of the cosmid contig.

To complement the phenotype of nim1 plants, transformation of the plants with the wild NIM1 gene is required. This can be achieved by transforming these plants with a cosmid containing this gene. To this end, the cosmid contig of the NIM1 region is constructed. Because Arabidopsis is transformed using AgRobaeterium. the cosmid vector is used as a binary vector.

DNA was isolated from BAC-04, BAC-06 and P1-18 and used to perform partial digestion using the restriction enzyme Sau3Al. 20-25 kb fragments were isolated using a sucrose gradient, pooled and filled in with dATP and dGTP. The binary vector (04541) was digested with Xhol and filled in with dCTP and dTTP. The fragments were then ligated into the vector. The ligation mixture was packaged and transduced into E. coli.

The cosmid library was screened with clones BAC-04, BAC-06 and P1-18 and positive clones were isolated. These cosmids were then examined by AFLP fingerprinting and arranged in a contigu of overlapping clones overlapping the NIM1 region. The sizes of the cosmid inserts were determined and limited restriction mapping was performed. The results are shown in Figure 10.

Example 2

Identification of a clone containing the NIM1 gene.

1. Complementation with stable transformation.

Cosmids that are generated from clones that include the NIM1 region (described above) are introduced into the Agrobacterium by tri-parent crosses. These cosmids are then used to transform Arabidopsis by vacuum infiltration (Mindrinos et al., Cell 78, 1089-1099) or by standard root transformation.

Seeds are harvested from these plants and allowed to germinate on agar plates with kanamycin (or other suitable antibiotic) as selection agent. Only plants that are transformed with cosmid DNA can detoxify the selection agent and survive. Seedlings that survived the selection were transferred to soil and tested for phenotype before or their offspring were tested for phenotype with nim. Transformed plants that no longer have a phenotype before identifying a cosmid (s) with a functional NIM1 gene.

2. Complementation in the transient expression system.

The ability of DNA clones to complement mutations with them is tested in two transient expression systems.

In the first arrangement, Arabidopsis plants bearing the PR1-luciferase (PR1-lux) transgene are used as recipients in bombardment. These plants are generated by transforming Columbia ecotype plants with the PR1-lux construct by vacuum infiltration followed by kanamycin selection of harvested seeds as described above. Transformed plants that express luciferase activity upon INA induction are self-pollinated and homozygous plants are generated. They are crossed with nim1 plants. In the transient assay, progeny plants of this cross that are homozygous for nim1 and PR1-lux were used to identify DNA clones that might complement the nim1 phenotype. For this, plants are first treated with INA as described in Example 1.1 above. After two days, these plants were harvested, surface sterilized, and plated on GM agar medium. Leaf tissue was then bombarded with cosmid, P1 or BAC clones (or subclones) from the NIM1 region, and leaf luciferase activity was determined after one day. Clones that induce luciferase activity contain the NIM1 gene.

In a second arrangement, plants are treated with INA as described in Example 1.1 above) and after two days then bombarded with cloned DNA (cosmid, BAC and / or YAC clones or subclones) from the NIM1 region and reporter plasmid. The reporter plasmid contains the luciferase gene under the control of the Arabiopsis PR1 promoter (PR1-lux). In nim1 plants, INA does not activate the PR1 promoter (as described in Example 1.2 above) and therefore cannot induce luciferase activity from the reporter plasmid. However, if the cotransformed clone contains the complementing NIM1 gene, INA induces the PR1 promoter as seen as induction of luciferase activity. Whole plants luciferase activity is determined one day after co-bombardment. DNA clones (clones or cosmid subclones, P1 or BAC) that induce luciferase activity significantly above background level contain the NIM1 gene.

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3. Changes in transcripts in lines with the nim1 phenotype.

Since plants with the nim1 phenotype have mutations in the NIM1 gene, it can be assumed that in some lines the gene is altered such that no mRNA is transcribed, or that abnormal (size) mRNA is produced. To verify this, an analysis of the RNa filters in nim1 lines was performed.

RNA was isolated from the Ws and Ler plants of these lines (after treatment with water or INA or BTH) and used to prepare Northern filters. These filters are hybridized to DNA fragments isolated from NIM1 locus DNA contig clones. The DNA fragments that identify nim1 lines with impaired RNA expression (altered size or concentration) are likely to identify part of the NIM1 gene. The DNA fragment and the surrounding DNA is sequenced and used to isolate cDNA (by library screening or reverse transcription PCR) which is also sequenced. The clone from which the fragment or the isolated cDNA was isolated is used to demonstrate the complementation of the phenotype with them in the stable and transient expression systems.

Example 3

Determination of the DNA sequence of the NIM1 gene.

1. Genomic sequencing.

Genomic clones that may contain the NIM2 gene are sequenced using methods known in the art. They include BAC-04, PI-18 and cosmids from the NIM1 region. For example, cosmids are digested with restriction enzymes and fragments that derive from the insert are cloned into a generally useful vector such as pUC18 or Bluescript. The larger clones of P1 and Bac are randomly fragmented and the segments are cloned in a generally useful vector. Fragments in these vectors are sequenced using conventional methods (e.g. by primer treading or generating insert deletions). The resulting sequences are assembled in a continuous sequence.

The insert sequence of the complementing clone contains the N1M1 gene. The approximate start and end of the NIM1 gene is deduced from the DNA sequence, sequence motifs such as TATA, open reading frames present in the sequence, codon usage, cosmid complementation data, relative positions of AFLP markers, and other relevant data collected (see Example 4, below). ).

4. cDNA sequencing.

The cosmid (s) or larger clones that contain the NIM1 gene (as described in Example 2 above) are used for cDNA isolation. This is achieved by using clones (or DNA fragments) as probes in screening a wild-type Arabidopsis cDNA library. cDNAs that are isolated are sequenced as described in cosmid sequencing and used in complementation assays. For this, the full-length cDNAs are cloned into an appropriate expression vector, downstream of the constitutive promoter. These constructs are used in the transient assays as described above. Alternatively, the cDNAs are cloned into a binary expression vector that allows expression in plant tissues and Agrobacterium-mediated transformation of the plants as described in Example 2 above. cDNA that contains the NIM1 gene (as determined by complementation, isolation with a closely linked AFLP marker, isolation with a cosmid fragment, or other deduction) is sequenced;

Genes from Ws-O and nim plants are isolated and sequenced. The genes are obtained from the cosmid of the cDNA library using a fragment of the isolated NIM1 gene as a probe. Alternatively, the genes or cDNAs are isolated by PCR using the NIM1 gene specific primers and genomic DNA or cDNA as template. Similarly, the them alleles from other nim1 lines (see Example 1.1 above) are isolated and sequenced in a similar manner.

Example 4

Description of the NIM1 gene and deduced protein sequence.

The sequence of the Iub gene of the NIM1 cDNA was determined as described in Example 3 above. The sequence was analyzed using DNA analysis programs such as are found from Genetics Computer Group (GCG), Sequencer or Staden packages, or any other similar DNA analysis program package.

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Specifically, the start and end of a gene are determined based on open reading frame analysis, presence of stop codons and potential start codons, presence of potential promoter motifs (such as the TATA sequence), presence of polyadenylation signals, and the like.

Also, the amino acid sequence is deduced from the open reading frame. Both DNA sequence and proteins are used to search databases for sequences with homologues such as transcription factors, enzymes or motifs from such genes or proteins.

Example 5

Isolation of homologues of the NIMI gene.

The Arabidopsis NIMI gene can be used as a probe in low-stress screening of a genomic or cDNA library to isolate NIMI homologs from other plant species. Alternatively, this can be done by PCR amplification, using primers designed from the sequence of the Arabidopsis gene and using genomic DNA or cDNA as template. The NIMI gene can be isolated from corn, wheat, rice, barley, rapeseed, sugar beet, potato, tomato, bean, cucumber, grape, tobacco, and other crops of interest and sequenced. Given the set of NIMI homologs sequences, new primers can be designed based on conserved gene sequences to isolate NIMI homologues from more distant plant species by PCR amplification.

Example 6

Niml-1 gene complementation with genomic fragments.

1. Construction of the cosmid contig.

The cosmid contig of the NIMI region was constructed using CsCl purified DNA from BAC04, BAC06 and P1-18. The DNA of the three clones was mixed in equimolar amounts and partially digested with the restriction enzyme SauSA. 20-25 kb fragments were isolated using a sucrose gradient, pooled and filled in with dATP and dGTP. Plasmid pCLD04541 was used as cosmid T-DNA vector. This plasmid contains a broad host range pRK290 based replicon, a tetracycline resistance gene for selection in bacteria, and a nptII gene for selection in plants. The vector was digested with Xhol and filled in with dCTP and dTTP. The prepared fragments were then ligated into the vector. The ligation mixture was packaged and transduced into E. coli XL-1 blue MR strain (Stratagene). The resulting transformants were screened by hybridization to clones BAC04, BAC06 and P1-18 and positive clones were isolated. Cosmid DNA was isolated from these clones and template DNA was prepared using EC EcoRI / Msel and HindIII / Msel. The resulting fingerprint patterns of the AFLP fragments were analyzed to establish the order of cosmid clones. A set of 15 overlapping cosmids spanning the region of n1 was selected (Figure 13). DNA cosmids were digested with EcoRI, Pstl, BssHI and SgrAI. This allowed the size of the cosmid inserts to be estimated and the overlaps between different cosmids as determined by AFLP fingerprints to be checked.

2. Identification of a clone containing the NIMI gene.

Cosmids generated from clones spanning the NIMI region were transferred to Agrobacterium tumefaciens AGL-1 by conjugative transfer in tripartite crossing with the helper strain Hb 101 (pRK2013). These cosmids were then used to transform the kanamycin sensitive nim-1 Arabidopsis line by vacuum infiltration (Mindrinos et al., 1994, Cell 78, 1089-1099). Seeds from the infiltrated plants were harvested and allowed to germinate on GM agar plates containing 50 mg / ml kanamycin as selection agent. Only plants that are transformed with cosmid DNA can detoxify the selection factor and survive. Seedlings surviving selection were transferred to soil approximately two weeks after sowing and tested for phenotype as described below. Transformed plants that no longer have a phenotype identify the cosmid (s) with an active NIMI gene.

3. Testing the phenotype with them of transtorms;

The plants transferred to the soil were grown in a phytotron for about one week after transfer. 300 pM of INA was applied as a fine mist to completely cover the plants using the sprayer. After two days, leaves were collected for RNA extraction and PR-1 expression analysis. On

187,851 plants were then sprayed with Peronospora parasChCH (EmWa isolate) and grown under high humidity in a growth chamber at temperatures of 19 ° C day / 17 ° C night and an 8 h light / l 8 h dark cycle. Eight to ten days after fungus inoculation, the plants were scored positive or negative for fungal growth. Ws and nim1 plants treated in the same manner were controls for each experiment.

Total RNA was extracted from harvested tissue using LiCl / phenol extraction buffer (Vcrwocrd et al., NAR 17: 2362). RNA samples were run on formaldehyde gels and transferred to GeneScreen Plus membranes (DuPont). Hybrydyzowaen filter LP cDNA probe-1 labeled with ³² P. After hybridization, the filters were exposed on plates to determine which traesformaety are capable of inducing the expression of PR-1 in the timing LNA. The results are summarized in Table 16.

Table 16 shows the complementation of the nim1 phenotype by cosmid clones.

Table 16

The name of the clone # transformants # plants with PR-1 ind. by INA / total number of plants tested (%) A8 3 0/3 (0%) A11 8 4/18 (22%) C2 10 1/10 (10%) C7 33 1/31 (3%) D2 81 4/49 (8%) D5 6 5/6 (83%) El 10 10/10 (100%) D7 129 36/36 (100%) E8 9 0/9 (0%) F12 9 0/6 (0%) E6 1 0/1 (0%) E7 34 0/4 (5) WS-control (wild) ON 28/28 (100%) control of the nim-1 phenotype ON 0/34 (0%)

NA-Not applicable

Example 7

Sequence of the 9.9 kb area of the NlM1 gene.

BAC04 DNA (25 µg, obtained from KeyGene) was the source of DNA used for sequence analysis. This BAC has been shown to be a clone that completely covers the region that complements the nimb mutants

DNA was randomly fragmented using the Cold Spring Harbor approach. Briefly, BAC DNA was fragmented in a nebulizer to an average molecular weight of about 2 kb. Fragmen187 851 ends were repaired using a two-step protocol with dNTPs, a polymerase. DNA from T4 and a Klenow fragment (Boehringer). The end-repaired DNA was electrophoresed on 1% low melting point agarose and an area between 1.3 kb and 2.0 kb was cut from the gel. DNA was isolated from the gel fragment using the freeze-compression method. The DNA was then mixed with EcoRV digested pBRKanF4 and ligated overnight at 4 ° C. pBRKanF4 is a derivative of pBRKanF1 that was obtained from Kolavi Bhat at Vanderbilt University (Bhat, KS, Gene 134 (1), 83-87 (1993). E. coli DH5a strain was transformed with the ligation mixture and the transformation mixture was plated on plates containing kanamycin and X -gal. 1600 white or light blue KanR colonies were selected for plasmid isolation Single colonies were picked into 96 well plates (Polyfilttronics, # U508) containing 1.5 ml TB + Kan (50 pg / ml). The plates were covered and placed on a rotating shaker. at 37 ° C for 16 hours Plasmid DNA was isolated using the Wizard Plus Miniprep 9600 kit (Promega, # A7000) according to the manufacturer's instructions.

Plasmids were sequenced using Dye Terminator chemistry (Applied BioSystems PRISM Dye Terminator Cycle Sequencing Ready Kit, P / N 402078) and primers designed to sequence both plasmid strands. Data was collected on ABI 377 sequencers. About 75% of these reactions gave useful sequence information. Sequences were edited and assembled into contigs using Sequencher 3.0 (Gene Codes Corporation), Staden gap4 (Roger Staden, email rs@mrc-imb.cam.ac.uk) and PHRED (Phil Green, email phg@u.washington .edu). The largest contig (approximately 76 kb) covered a complementary area with an average coverage of 7 independent counts / base.

An area of about 9.9 kb defined by the overlapping cosmids El and D7 was identified by complementation analysis to constrain the nim1 region. Primers that flanked the vector insertion site and specific for the backbone were developed using Oligo 5.0 Primer Analysis Software (National Biosciences Inc). DNA was isolated from cosmids D7 and El using modifications to the ammonium acetate method (Traynot, P.L., 1990, BioTechniques 9 (6): 676). This DNA was directly sequenced using DNA Terminator chemistry as above. The obtained sequence allowed for the determination of the endpoints of the complementary area.

A truncated version of the BamHI-EcoRI fragment was also constructed, resulting in a construct that did not contain the "Gen 3" region at all (Figure 13). The following approach was necessary due to the presence of HindIII sites in the Bam-Spe DNA region. The BamHI-EcoRI construct was digested completely with Spel and then split into two separate double digest reactions. One sample was digested with BamHI, the other with HindIII. A BamHI-Spel fragment of 2816 bp and a HindIII-Spel fragment of 1588 bp were isolated from agarose gels (QiaQuick Gel Extraction kit) and ligated with BamHI-HindIII digested pSGCG1. DH5a was transformed with the ligation mixture. Resulting colonies were screened for the correct insert by digestion with HindIII after dNa preparation using Wizard Magie MiniPreps (Promega). A clone containing the correct construct was electroporated into the Agrobacterium GV3101 strain for transformation of Arabidopsis plants.

Example 8

Identification of the NIM 1 gene region by allele sequencing.

Table 17

Genetic segregation of mutants with non-inducible resistance

Mutant Generation Female Wild male nim1 nim1-1 ^a F1 wild ^b nim1-1 24 0 F2 98 32 nim1-2 F1 nim1-2 wild 3 0 nim1-3 F1 nim1-3 wild 3 0 nim1-4 F1 nim1-4 wild 3 0 nim1-5 F1 nim1-5 wild 3 35

Phenotype

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c.d. table 17

him 1-6 F1 him 1-6 wild 3 0 nim1-2 F1 him 1-2 niml-1 0 15 him 1-3 F1 him 1-3 niml-1 0 10 him 1-4 F1 him 1-4 niml-1 0 15 him 1-5 F1 him 1-5 niml-1 0 15 F2 9 85 him 1-6 F1 him 1-6 niml-1 0 12

"Data from Delaney et al. (1995) PNAS 92.6602-6606 ^b Wild type means the Ws-O strain

1. Genetic analysis.

In order to establish the dominance of various mutants showing the nim1 phenotype, pollen from wild plants was transferred to the nim1, -2, -3, -4, -5, -6 stigma. If the mutation is dominant then the nim1 phenotype will be observed in the resulting F1 plants. If the mutation is recessive, the resultant F1 plants will show a wild phenotype.

The data presented in Table 17 shows that when niml-1, -2, -3, -4, -6 are crossed with the wild type, the resulting F1 exhibits a wild type phenotype. So these mutations are recessive. In contrast, the progeny of the nim1-5x wildtype in F1 all show the nim1 phenotype, indicating that it is a dominant mutation. After INA treatment, no P. prasitica sporulation was found on wild plants while the F1 plant maintained growth and some P. parasitica sporulation. However, the phenotype of them in these F1 plants was less severe than that observed when mim 1-5 was homozygous.

To determine allelicity, pollen from kanamycinomate mutant niml-1 plants was transferred to the nim1-2, -3, -4, -5, -6 stigma. Seeds resulting from the cross were sown on Murashige-Skoog B5 plates containing kanamycin at 25 pg / ml to check for hybrid seed origin. Kanamycin (F1) resistant plants were transferred to soil and examined for the nim1 phenotype. Since the F1 progeny of the cross of the nim 1-5 mutant with wild-type Ws showed the phenotype of nim1-5xnim1-1.

As shown in Table 17, all resulting plants showed the nim1-1 phenotype. Thus, the mutation in nim1-2, -3, -4, -5, -6 was not complemented by nim1-1; these plants all belong to the same complementation group and are therefore allelic. Analysis of the F2 progeny from the nim1-5xnim1-1 cross also showed a nim1 phenotype, confirming that nim 1-5 is the nim1 allele.

2. Sequence analysis and subcloning of the NIM1 region.

The 9.9 kb region containing the N1M1 region was analyzed for the presence of open reading frames in all six frames using Seguencher 3.0 and a GCG packet. Four regions containing large ORFs were identified as possible genes (gene regions 1-4). These four regions were amplified PCR from wild-type parent DNA and six different nim1 allelic variants. Primers for these amplifications were selected using Oligo 5.0 (National Biosciences, Inc.) and synthesized at Integrated DNA Technologies, Inc. The PCR products were separated on 1.0% agarose gels and purified using the QIAQuick Gel Extraction Kit. The purified genomic PCR products were sequenced directly using the primers used for the initial amplification and with additional primers designed for sequencing through any regions not covered by the original primers. Average coverage for these genes was approximately 3.5 reads / base.

Sequences were edited and assembled using Sequencher 3.0. Base changes specific to different nim1 alleles were identified only in the region designated as the Gene 2 region.

The items listed in Table 18 relate to Figure 14 and relate to the upper thread of the area

9.9 kb given in Figure 13. The open reading frames from the regions of the genes described in Figure 13 as 1, 2, 3 and 4 were sequenced and the changes in the various alleles thereto are shown in the Table. The variations which are described in the upper thread 5c to 3c in figure 13.

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You can see that the NIM1 gene has been cloned and that it lies in the Gene 2 region because there are amino acid changes or sequence changes in the open reading frame of the gene 2 region in all 6 alleles of it. At the same time, at least one of the nim1 alleles shows no changes in the open reading frame within regions of gene 1, 3 and 4. Thus, the only gene within region

The 9.9 kb that may be NINM1 is the region of the gene 2, genNIM1.

Part of Table 18 shows changes in the Arabidopsis Ws ecotype relative to the Columbia Arabidopsis ecotype. Figures 13, 14, 15 and all others relate to the Columbia Arabidopsis ecotype which contains the wild type gene in the experiments that were performed. Changes are listed as amino acid changes in the NIM1 gene 2 or region and are given as base pair changes in the remaining regions.

Figure 13 shows the various panels that describe the cloning of the NIM1 gene and describe the entire 9.9 kb area. Figure 14 is the sequence of the entire 9.9 kb region in the same orientation as described in Figure 13. Figure 15 is a sequence of a specific region of the NFM1 gene which is the gene 2 region shown in Figure 13, the sequence of Figure 15 contains the NIM1 gene. Figure 15 shows the amino acid sequence in the single letter code and shows the full length cDNA and RACE product that was obtained in capital letters in the DNA sequence. Some of the allele mutations found are shown above the DNA sequence and the n and 1 allele where this change occurred is indicated.

Sequence analysis of this region and sequencing of the various nim1 alleles (see below) identified the region of the cosmid that contains the nim1 gene. This region is defined by a BamHI-EeoRV restriction fragment of approximately 5.3 kb. Cosmid DNA from D7 and plasmid DNA from pBluescript (pBSII) was digested with BamHI and EcoRV (NEB). The 5.3 kb D7 fragment was isolated from agarose gels and purified using the QIAquick gel extraction kit (# 28796, Quiagen). The fragment was ligated overnight with Bam-EeoRV digested pBSII and the ligation mixture was transformed into E. coli strain DH5a. Colonies containing the insert were selected, DNA was isolated and confirmed by digestion with HindIII. The BamEcoRV fRagment was then inserted into a binary vector (pSGSG01) for transformation into Arabidopsis.

3. Northern blot analysis of the four gene regions.

Identical Northern filters were prepared from RNA samples isolated from the Ws and nim1 lines treated with water, SA, BTH and INA as previously described (Delaney et al., 1995, PNAS 92, 6602-6606). These filters were hybridized with PCR products generated from four gene regions identified in the 9.9 kb region of the NIM1 gene. Only the gene region containing the NIM1 gene (gene region 2) had detectable hybridization with the RNA samples, indicating that only the NIM1 region contains the detectable transcribed gene (Figure 16 and Table 18).

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Table 18 shows the changes in the gene allele sequences.

Table 18

Genoa area Allele / ecotype 1 (rules 590-1090) 2 (PTT1) (rules 1380-4010) 3 (rules 5870-6480) 4 (rules 8140-9210) nim1-1 no change t inserted in 2981: 7AA alteration and premature protein termination no change no change nim1-2 no change g na a in 2799: His na Tyr no change no change him 1-3 no change t deletion at 3261: 10AA alteration and premature protein termination no change no change nim1-4 no change c to t in 2402: Arg to lys no change no change him 1-5 no change c to t in 2402: Arg to lys no change no change nim1-6 g to a in 734: asp to lys g to a in 2670: Gln to Stop no change no change WS (compared with Columbia) no change a on gw 1607: Ile na Leu a on cw 2344: intron t on gw 2480: Gln na Pro g na cw 2894: Cheese on Trp ggc deletion in 3449: loss Ala c on tw 3490: AJa on Thr c on tw 3498: Cheese na Asn a na tw 3873: non-coding g na aw 3992: non-coding g na aw 4026: non-coding g na aw 4061: non-coding t na a w 5746 a natw 5751 t na a w 5754 c to t w 6728 a na t w 6815 tna c w 6816 t per g in 8705 g per t in 8729 g per t in 8739 g per t in 8784 c per a w 8789 c per t in 8812 a per g in 8829 t per g in 8856 a per c in 9004 a per t in 9011 a per g in 8461 RNA detected No Yes No No

The items listed in the table refer to Figure 14 having a 9.9 kb sequence. All the nim1-1 through nim1-6 alleles are from strain WS. Columbia-0 represents the wild type.

We have also shown that the gene region 2 (Figure 13) contains the ίueecjoe, alnc NlM1 gene by performing additional complementation experiments. A genomic BamHI / HindIII DNA fragment containing the gene region 2 was isolated from cosmid D7 and cloned into the binary vector pSGSG01 containing the kanama resistance gene ^^ (Figure 13, Steve Goff, oral communication). The resulting plasmid was transformed into Agrobacterium strain GV3101 and positive colonies were selected on kaeamcccnle. PCR was used to verify that the selected colony contained the plasmid. Eaeamizin susceptible plants were 1-1 infiltrated with these bacteria as previously described. The resulting seeds were harvested and plated on GM agar containing 50 pg / ml

Kanamycin 187 851. Plants surviving by selection were transferred to soil and tested for complementation. The transformed Ws control plants and plants were sprayed with 300 pM INA. After two days, leaves were collected for DNA extraction and PR-1 expression analysis. Plants were then sprayed with Peronospora parasitica (EmWa isolate) and grown as previously described. Ten days after fungal infection, plants were scored and scored positive or negative for fungal growth. All 15 transformed plants as well as the Ws controls were negative for fungal growth after INA treatment, while the controls were positive for fungal growth. RNA was extracted and analyzed as described above for these transformants and controls. The Ws controls and all 15 transformants showed induction of the PR1 gene after INA treatment, while the controls showed no PR-1 induction by INA.

4. Isolation of the NIMI cDNA.

An Arabidopsis cDNA library prepared in the IYES expression vector (Ellege et al., 1991, PNAS 88, 1731-1735) was plated and plaque replicas were made. The filters were hybridized with ³² P-labeled PCR product generated from the gene region containing them. 14 positive clones were identified from a screen of approximately 150,000 plaques. Each plaque was purified and plasmid DNA was recovered. The cDNA inserts from the vector were digested with EcoRI, purified by agarose gel electrophoresis and sequenced. The sequence obtained from the longest cDNA is shown in Figure 15. To confirm that we had obtained the 5 cDNA end, the Gibco BRL to 5'RACE kit was used according to the manufacturer's instructions. The resulting RACE products were sequenced and found to contain the additional bases shown in Figure 15. The transcribed area present in both cDNA clones and detected in RACE is shown in upper case in Figure 15. Allele changes are shown above the DNA strand. Capital letters will indicate the presence of a cDNA Or clone in the sequence detected after RACE PCR.

Example 9

Characteristics of the NIMI gene.

Multiple sequence comparisons were performed using Clustal V (Higgins, Desmond G. and Paul M. Sharp (1989). Fast and sensitive multiple sequence alignments on a microcomputer, CABIOS 5: 151-153) as part of DNA * (1228 South Park Street, Madison , Wisconsin, 53715) of the Lasergene Biocomputing Software for Macintosh (1994).

Certain regions of the NIMI protein were found to be homologous in the amino acid sequence to 4 different rice cDNA products. Homologies were determined using NIMI sequences in GeneBank BLAST searches. Comparisons of the NIMI homology regions and the rice cDNA products are shown in Figure 19. The NIMI protein fragments show 36 to 48% of the amino acid sequences identical to the 4 rice products.

Example 10

Phenotypic characteristics of various alleles nim 1.

Analyze Rlllll's responses to chemical solvents.

We analyzed the differences between the various alleles in terms of chemical induction of PppR gene expression and resistance to Peronospora parasitica (see figures 17 and 18). Mutant plants were treated with chemical inducers and then tested for PR gene expression and disease resistance.

2. Plant growth and the use of chemicals.

Wild-type seeds and seeds of each of the alleles (niml-1, -2, -3, -4, -5, -6) were sown on MetroMix300 media, covered with a clear glass dome and placed in the dark at 4 ° C for 3 days. After 3 days at 4 ° C, the plants were transferred to the phytotron for 2 weeks. Approximately 2 weeks after sowing, the selected seedlings produced 4 true leaves. The plants were then treated with H ₂ O.5mM SA, 300 µM BTH or 300 µM INA. The chemicals were applied as a fine mist to completely cover the seedlings using a sprayer. The water-treated control plants were returned to the growth phytotrone while the treated plants were housed in an identical but separate phytotrone. After 3 days, the plants were divided into 2 groups. One group was collected for RNA extraction and analysis. The second group was inoculated with P. parasitica.

3. Inoculation and analysis of Peronospora parasitica.

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The P. parasitica 'EmWa' isolate is a P.p. isolate that is compatible on the Ws ecotype. Compatible isolates are those that are capable of causing disease in a given host. P. parasitica "NoCo" isolate is incompatible with the Ws but compatible with the Columbia ecotype. Incompatible pathogens are recognized by the potential host, causing the host to respond which prevents the development of the disease. Three days after treatment with chemicals, plants treated with water and chemicals were inoculated with the compatible isolate "EmWa". The "NaCo" inoculation was performed only on water-treated plants. After inoculation, the plants were covered with a clear plastic dome to maintain the high humidity required for successful P. parasitica infection, and placed in a growth chamber at 19 ° C day / 17 ° C night and 8 hr light / 16 hr dark cycles.

Plants were analyzed microscopically at various time points after inoculation to assess symptom development. Under magnification, fungal sporulation can be observed in the very early stages of the disease. The percentage of plants / pot showing sporulation after 5 days, 6 days, 7 days, 11 days and 14 days after inoculation was assessed and the sporulation density was also recorded.

Figure 18 shows the disease assessment of the various alleles with them after inoculation with P. parasitica. The most distinguishing time points are 5 and 6 days post vaccination. At 5 days after inoculation with him, 1-4 shows 80% infection with all chemical treatments performed, showing that this allele / genotype has the most severe susceptibility to disease. At 6 days after inoculation with them, -2, -4, and -6 show significant disease incidence after all inducing chemicals. However, nim 1-5 shows less infection than wild-type Ws after all treatments on Day 6. Thus, niml-5 is the most resistant to disease of all the alleles. niml-2 appears to be intermediate in sensitivity to BTH but not to other inducing chemicals.

Expression of the PR-1 gene indicates that nim 1-4 is the least responsive to all the inducing chemicals tested (Figure 17) while nim 1-5 shows elevated levels of PR-1 expression in the absence of inducers. The results on the expression of the PR-1 gene are in agreement with the disease assessment performed with P. parasitica (Figure 18) and demonstrate that alleles thereof can confer resistance or sensitivity.

The samples obtained above were used to analyze the expression of the NIMI gene (Figure 17). In wild-type plants, mRNANIM1 was present in untreated controls. After treatment with SA, INA, BTH, or infection with a compatible pathogen, mRNANIM1 accumulated to higher levels. Differences in mRNA levels for NIMI were observed in the alleles of them compared to the wild type. The amount of NIMI mRNA in the untreated mutant plants was lower than that observed in the wild-type with the exception of niml-2 and -5, where the amounts were similar, and 1-1, -3 and -4 had low levels of NIMI mRNA while nim 1-6 had very low accumulation of NIMI mRNA. An increase in NIMI mRNA after SA, INA or BTH was observed in niml-1, -2, -3 but not niml-5 or -6. However, this increase was less than that of wild-type plants. After infection with the pathogen, additional bands hybridizing with the NIMI cDNA probe were observed in both wild-type and mutants, and NIMI mRNA levels were elevated relative to untreated controls, except 1-6.

Figure 18 shows the assessment of disease resistance by assessing infection for various alleles with them as well as for NahG plants at various times after inoculation in Peronospora parasitica. WsWT indicates by the wild-type parental line Ws in which the Ws alleles are found. The various alleles with them are shown in the table and the NahG plant is also shown. The NahG plant has been previously published (Delaney et al., Science 266, pp. 1247-1250) (1994). Arabidopsis NahG is also described in WO 95/19443.

The NahG gene is a gene from Pseudomonas putida that converts salicylic acid into catechol, thus eliminating the accumulation of salicylic acid, a necessary transduction signal for SAR in plants.

Thus, Arabidopsis NahG plants do not show normal SAR. Additionally, they show a much greater overall sensitivity to pathogens. Thus, NahG plants serve as a genus

187 851 for universal sensitivity control. Additionally, NahG plants are still responsive to the chemical inducers of INA and BTH, this is shown in the lower two panels in figure 17.

In Figure 18 it can be seen that the alleles n and m 1-6 appear to be the most acute, this is most easily seen at the earlier time points, described earlier in the Results section herein, and from the results presented in the EmWaBTH panel, lowermost panel, in the figure. Additionally, the nim 1-5 allele shows the greatest response to both INA and BTH and thus is the weakest allele of both.

NahG plants show a very good response to INA and BTH and look very similar to the nim 1-5 allele. However, at the late time points, Day 11 in the figure, disease resistance induced in NahG plants begins to decline and there is a major difference between INA and BTH as INA-induced resistance drops much faster and more sharply than BTH-induced resistance in NahG plants It is also seen from these experiments that INA and BTH induce very good resistance in Ws to EmWa and the niml-1, niml-2 and other nim2 alleles show essentially no response to SA or INA regarding disease resistance.

Figure 18 lists the percentage of plants that show sporulation after infection with the EmWa R parasitica race, and each histogram indicates the number of days post-infection when the infection is assessed.

Northern blot analysis to analyze PR1 SAR gene expression was also performed on the same samples as shown in figure 17. Figure 17 shows that the wild-type plant shows a very good response to salicylate, INA, BTH and also to pathogen infection as manifested by increased expressing the PR1 gene. The niml-1 allele, on the other hand, shows only a very limited response to all chemical inducers, including the pathogen.

Pathogen induction is at least several times lower in the niml-1 allele than in the wild-type. The niml-2 alleles, nim 1-3 and nim 1-6 show a response similar to the niml-1 allele to the different treatments. However, the nim 1-4 allele is essentially not expressed in response to any of the inducers used. Basically, only the background level is observed. The niml-5 allele exhibits a very high background level relative to the water control and this background level is maintained with all treatments, however there is limited or no induction by chemical inducers.

NahG plants are a good control showing that they are unable to induce PR-1 in the presence of SA, on the other hand INA and BTH both induce very strong expression of PR1 at high levels. The effect of pathogen infection is similar to that of SA; there is no PR-1 expression in EmWa treated NahG plants.

The same RNA samples produced in the infectious studies were also screened with the NIMI gene using the full-length cDNA clone as a probe. Figure 16 shows that INA induces the NIM2 gene in the wild-type Ws allele. However, the niml-1 mutation allele shows a lower basal expression of the NIMI gene and is not inducible by INA. This is similar to what is observed for the niml-3 allele and the niml-6 allele. The nim 1-2 allele shows approximately normal levels in the untreated sample and shows similar induction to wild type as does the nim 1-4 allele. The nim 1-5 allele seems to show a higher level of basal NIMI gene expression and much stronger expression when induced by chemical inducers.

Induction of NIMI by resistance chemical inducers and other inducers is consistent with its role in defense against pathogenesis and is further evidence that we obtained the correct gene in the 9.9 kb region.

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Sequence list (1) GENERAL INFORMATION:

(i) APPLICANTS:

(A) NAME: Novartis AG (B) STREET: Schwarzwaldallee 215 (C) CITY: Basel (E) COUNTRY: Switzerland (F) ZIP CODE (ZIP): 4002 (G) PHONE: +41 61 69 11 11 (H) TELEFAX: +41 61 696 79 76 (i) TELEX: 962 991 (ii) TITLE OF THE INVENTION: GEN GIVING RESISTANCE TO

PLANT DISEASES AND ITS APPLICATIONS (iii) NUMBER OF SEQUENCES: 11 (iv) METHOD OF COMPUTER WRITING:

(A) ENVIRONMENT: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentin Release # 1.0, Yersion # 1.30 (2) INFORMATION FOR SEQ. ID NO: 1:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 9,919 base pairs (B) TYPE: Nucleic acid (C) STRAND: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTISENSIVE: NO ( xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 1:

TCATGATGAA TTCOGTGIAG GGllGlGiTi 'TAAAGAIAGS GATGAGCTGA AGAAG3O3GT

GGACTGCTGT TCCATLAGAG GGCAGCAAAA GIGIGEAGTA CAAGAGATIG AGAAGGACGA. 120

187 851

GIATCWUnT AAATOOATCA GAIOGAATG CAMTGTOG CETCG33CAG ATICAA1AGA 180

AGACATOGA CUGHAAGA TAACIAGIG TAGUGICC ACMACnOT IGITCIATIA 240

AGCCGAAAA CTTCAACTG TAATTTGAG CCAGAGA3TT TGAGT3TCTG ATCAGGGTAC 300

AACOCACTCT AACAGCAG TTGAAAAGTT TGGT3ACITO CTTAAAftCTT CAAAGCT3C3 360

GGCCGCAGAA CAGGAGIA TCAAAGATCA GAgTTICAGA GTATT3OCIA AACTAATT3G 420

CIGCATTTCA CTCTCIAAT GGGCIACTIG TGGACTGCA TATCAGCTTT TCCCIAATCC 480

TGAATTTGCA TCCITCGGIG GCOGTITIG GG03ITIOCCA CAGTCCAIIG AAGGGTITCA 540

ACACTIAGA CCTCIGATCA TAGTOGAIC AAAAGACTIG AA2GGCAAGT ACCCTAT3AA 600

ATIGAIGATI TCCTCAGGAC TCGAOGCIGA T3AITGCOT TTCGOGCTTG CtCITICCGCT 660

TAGCAAA3AA GTGTOCACTG AAGTT33CG TTGGTTTCIC ACTAATATCA GAGAGAAGGT 720

AACACAAAGG AAAGACGHT GOCTCGTCTC CA3IOCTCAC COGAC3IAG TTGCUGTTAT 780

IAACGAACOC GGATCACTGT GGCAGAACC TTGGGICAI CACAGGTTCI GTCTGGATTG 40

TTITTGCTTA OCCTTOCCTO ATKUTTIOG AGACTACftAC CIGGTGC3CO TTGTGAAβOA 900

GGCI ^ GATOO COACGIOA3C AGAGATT ΊGCITCOΊCC AIAAGGACA TAAAAAGAA 960

GGACICAGAA GCICGGAAAT GCTTAGOCCA ATTOOOIOAA AATCAGTGGG OIOT ^ GOΓOA 1020

TGACCAGEGG TCCGACADA? GGAGTOATOA OGCIAGCCO AGAGATTTG AGGGCAATTT 1080

GT3AAA3CCT ΊOAίGICTOIT GCTOEAIOA3 TSACAGCSAA OGOAOCIGOC CATGTGGGAA 1140

GTTICftAICG AAGAAGTrTC CATOTATGCA OOOAGAAATG GT3CAAAGGA TTGTΊAA ^ OIT 1200

GTGTCAIT2A OAAAΊσΓTG3 ATOCAITOGA GOTGAOIAG AGATTGCACC TIAOAOGOOO 1260

ACTCAGIGIT C1CΊ1AIOΊO IGACCIGAA AOIACCIIGO T3T: SIAAIO GAGTTACA / AA 1320

AGGITAAAGG ACGACICCGIG CCGCAICOCIA TCCOCIGCAT GIKOOC3AC GTΊCCG! GGAA 1380

CTIGCAICII OIIITGG1TO I ^r [G3TGGC.CC CIATCOACOA GiTrGCCOCAT UGACAIIAI 1440

IICCCCOTn GATIOICGOA AOICI3IAAO ACOCI: OCT0G GTIITGTIG AK7IAOCICA 1500

CTCTCIATIA OACCICIGIA ICOOCIIGGT IOCCCIIGTI AOCAOCTTro TITCAAGOAC 1560

COOTGOC3OA CICATAOCCA GGAI ^ OCCCA OGCCGCGO3A CAOTCICTCC OGC! OCCOGCI 1620

AGCOCICACC CGTOCOCGIO CTOCIGCCAA CCGCAIICIA TOG'TAOAGCC ACCAIIAOCC 1680

187 851

TAAt3j33CAA GAGIOACE GA0®03AIG AffloAGITlA. 03317500 TCTITCCAOO 1740

OGITCMTTC GKD3K3fflAG AAGFO3AATC T3K5GG3AC GAATnOOIA ΑΤΓΟΟΑΑΑΠ ’1800

GTCCICACIA AAGOOCTICT T15GTGICI0 ΊΤ3Ι5! ΙΤΠΧ ATOEACCTIT GCTICTITIO 1860

Τ50ΙΌ3ΓΠ0 TCAGOOGT CGIUTIOOC ΟΏΑΟΟΟΑΟΓ TGfiGTCAAGT 00K5C5GIT 1920

C535AICIO3 T0®GC5CIG COaOGCG 0333AO & T 03ΓΠΌ003Α GTTOCACTGA 1980

Ί0Μ7 & ΑΑΑΑ AACAAG3TCA GACfiOCftAGT AAC5AAACCA ΤΟΓΠΆΑΑΞΑ TCATTEAGIT 2040

TIGITOTTG T3M5AGGRG TC0GAT3W3 T33GK5GAA T3CM5C033 TTTI5GAAAG 2100 ατητσο OTCTTKar GcrcnciSG gattctcpaa gstoceatct tooootg 2ieo

TCMGTICIC TTCGEACEJG T3AGAO33IC Α330ΙΌ3Ο CT5GK5CEA IGAACICACA 2220

1UITCCCTIC ΑΓΠ00303Α TCIOCR1TCC ΑΟΟΙΤ3ΙΟΟΓ T003TIGGAA AAAGA03ITG 2280

AGCAAGIGCA ACI5AAC5Gr Q3A03AC5CA AAGAATAGIT ΑΚ5Π50ΓΓ C5CTC5GTIT 2340

CCEAMAGAG AGGACKEAAA TTIAATTCAA ACATM5AGA AAEAAGAOT GMAGATAOC 2400

TCDOTITCA AGATO3AGCA G33K5TCTT CAAITC5TCE 03003303 C5AAAGAG3G 2460

AG3AACATCT CEAGGAATIT ΟΓΙΌΙΌ3ΓΓΤ CTCTTCTTOC ΤΟΠΟΑΠΤ CI5CAC5TAG 2520

TO33CCITIG AGAGAAIGCT IGOOTOCTC 033ffiIATI5 TI5CATTC5A 00303070 2580

ΟΟΟΠΟΓΠΤ G03ATC5T3A GIGOOTOT ACCTT3C5AA GTIO0ITCT3 ΑΤ33ΟΙΌ3 2640

ACCTTTIT02 ΑΑΊΑΟΟΟΆ GEATC5KIT3 Τ333Γ03ΠΟ G3C7OO3CAG GAACAT3AAG 2700

CAC03EAW ΟΟΟΟΙΌ33ΑΤ TOOEATGGIT (30TC03CA AGATC5AGIT ΠΜΑΑ0ΑΚ 2760

TCTIGOOOTC TTCACATTOC AA75T3C5AC AGOGAAAIGA AS05CA03 CATC5T0I5G 2820

ΑΊΤ33Κ7ΙΌΑ 1Ό3ΓΟΠΤ3Α AAAGC57OT SOAAGTC5. MATCAICOG AGTCAAGIOC 2880 ororamcA Troasor ghtciteac ttdoito tccaaaccaa goicitito 2940

TCI51CAAIT ATdCITIAA CAAGCICITC O33CAATCAC ITTTCAAGAC TAAO303OC 3000

TACATI5GAC TOC5M5A TCTCITEACA T3I5TCCAAT A00ITCM5C ΑΑΟΟΓΠΑΟΟ 3060

ACTOKEATEA GCAAGCTIGA GIMAACCAA TCIGICCTCT AT5ACAACIT TCICTACAAC 3120

GICCAM5AG TGCCTCTCAA ATACAAATAC AAGDOCAA GTAAGAAC5T ATTC5TCAAT 3180

GIGIAACCAT AGCTI5AT3C AGATC3TCIT TEAOCK305 GAGAGEAA1T AATTC5G3GA 3240

187 851

TdGAAGAT GAAAGCCAAA TAGAGAACOT CCAAdAA. ATOCAOCGCC GGCCGGCAAG 3300

CCCAGTTOGCA GCAATTCTCG TCT3CGOATT CAGftAAOTCC TnSGGGCGGC G3TCTCAOTC 3360

G3CTOOIGGCA AACATAAGCO AAAACAGTCA CAAOCGATC GAAACOGAOT TCGTAATCCT 3420

TCGCATCTC CTTAAG5CTC3 AGOTTCACGG GGGGOGTTGT GTTGGAOTCT TTCTOOTTCT 3480

TAGCGGOGGC TAAAGCGCTC TTGAAGAAAG AGCTTCTOGC TGACAAAACG CAOOCGI] TXGA 3540

AAGAAACTTC CCOUCCCTCG GAGAGAACAA GOTTAGOGC GOTGHA3AAA TCATCOGGCG 3600

AGTCAAAGAC GGATTCGAAG CGCTTGGAG OCATTGCAG AGGCAGATACA TCAGSTCCGG 3660

TGAGTACTTG TTCHCCG3CC AGATAAACAA TAGAGGAGTC G3TCTTATCG GTAGCGACGA 3720

AACTAGGCT GCTGATTTCA TAAGAATCHG CCGAATOCATC AATCGIIIGGG TCCATOACA 37 ^ 0

GGTCOGATG AATTGAAATT CACAAATTAA AGAGATCTCT GCTAATOAAO GAftUAGACCT 3040

TATCAACTGG ATTTGGTTAA AGATCGAA3A TTAACOATTGA CGAGCAGA3C CAAGTCAAGT 3900

CAACGAGAT ¹ GGTGGTGAG TATGAAGAAG CATOCTOGTC CCACGdTTTA CATTTCACCA 3960

AAACCGCTAA ATTTCCAGG AAGGATCTT TCK3GGAT CTTTTTTAAA AAGATATAAO 4020

AGGAAGCTAA AOCG (GITOIG GTTATAAATG TTAGTATTTA TACCGOAGC ATTTTUTT 4080

GCTAATTTTT GGTATATGAGA AGTTCATTOC Q3TTCUTAA GCCOCT3AAO CAAACCAGAT 4140

TTGGAGTGA TATAAATATA TAAAATTTAT TTTTCATCCG GITOGTTATT TTCATATAAA 4200

TATATAAATA TIATTTTTTA AATTTAAGAA TTAGATTTAC ATGTGAAACT TACATTTOTG 4260

TTTATTTTOT TTGAAGTAAA ATGATAAAGG GAACGm TAAGTTTCAT GOTTTATTCA 4320

CATAAGTTTT GTAATGTATA TT3TKTTTTT OgTTUAUGIA AAAAGTAATT TTCAGTCTTC 4380

AGCAUGTTTA CAOTADAATT AAATCAACTC GAATATTTCC TOUAACTATT CTCCTTOTTC 4444

TTAGOAAAT GAAAAO3CTC TTCACAACAA AATCATTATA GTHEGGAA. TAAATTACAT 4500

TAAAAACAH AAACTCATAA TOAATATATT TTTTTAATTA GGATTT3ATT TAAAAAOAAT 4560

TATTUATAO ATATAAAAGA CTTCTTTAGT TATTTOCCTT CAACTTCTCG TTCTOAATCA 4 ⁶ °

TOCGATAAAT CA3CTTTTTC AATAACTACG AGGTAAAAGC AAATTCATAA CACGTCCAAA 4680

CCAAlTTTGGC TCATCCTTCA CTTOATTOGT CTTTTCCGGA CTCGATOTTG CTGGAAACTI 4740

AGAAGAAGAA GGAATCTOCA TAATCACCTC TTGGTTCCTC AOOGCTGAO TCATTTTGTT 4800

187 851

GGATC3AAAA CG2AK33A3AT CAGAAAATCA. AAA3AEA3C3T TAAAGATGOC TATGAATACA 4860

ACAACCIAAG ATATETIOA. AIAAACAGAG TCTTIATAT AGGAGTAIA ATAAGGTAAA 4920

TAAAIATTTC CITIOCGCGI TTIITACTTT TCTATITCIT AAATGATAAG TIAAATIAGG 4980

ATAAGATITC TATCATITTA AGIAAm CAATAACTCT CTATAACTCA ATAGCATCAC 5040

ATATTEAATT AAITTIACTA ATTAICTIIT GAACAMITT AT3AAATAGT TITCTTUAA 5100

TTAATTTnT AAAATCATAT AITATTAAAAI TTAATTCAAT CAATCT3AIA. TAATTTTTTT 5100

ATCTTCTACC ATCTATTATA GTTCAIAAAT ATTCTGATAA ACTTTAGATA AACACOCCAT 5220

GCOAAAAT TTAATAAATT TIGIGTACGA TCCGTTTTTT TTGGAGAAIA. TATATACGT3 5280

GACAGCATAC CGTACATATA TTCTATAAAA GCTTATAAAA CATAGATACG GGTTATATIG 5344

GTAAGHATA AATAEAT3TA AACAATACTA AGATATTACG T3TTGTGTCT AAATATGTGT 5400

TGCTTTAGAT AITATGTATA TCTAAIATAT TAAAATATCT TTEAnAACT AATATATTAT 5460

TTAAGAGAGA AAATT3G3AC ACTAHTTCT ATACACTAC TCITITCAAC TATAAACAGG 5520

AACCTTGT ATAATAAAAT AACTAGCCAA AAAATCAGAT TAAATATTCA TAAAACAATC 5580

TTTGTATTA TTACATAAAC CIAAGAAACA AAATTCAATA TICCTIIIIA CCIIATAAAA 5044

AACAATTAAA CATCACIAGA TAUTTATTO CCCCACATTO AGCGAGCCAA TTGAGACTTC 5700

AGACTTGAGA TCCITCTCAA CICGHIGC ATTTGTCGGC CCATTIITT TATITTIITT 5760

TTAAAGTGIC GGCCCGTIOC TTCTTCOGTT CAGATCAACC CTCTCGTAT CAGAACAAAA 5820

CGGAAAACAA ACGAAAGAAC AATCAGATCC CTCTTITITI GCATAAACIA AAITCAACTT 5880

CTCTGC3TTT ATCTTGIAGA GGCAACCACG ATCACTACTA CGAAACAATA CAAOIKGTT 5940

GCUGGAGTC CAOGTAATCA AATCTACTCC AATCCTTTTA ATATCTTTCA CTTTAACCCA 0000

CGACTTTIGA AAACT3CTCT TIAAAACCCA TAACTCGIGA ACATCTTCTT GATCTITCTT 0000

TCTCCACTGA CGAATAGCAC CTACTTCCC TTCGTATCTI ACTAATCCIG AGAAAACATC 0120

AGAGTTCGGA GTAT3GAAGA AGGACCAAGT TTCGGTTTTC AGACAAAACC GGATCACATT 0180

GTTGTTCCGT GATATCCAAT GCAAGAACCC CGAAAidTGT ATCHGTIraG AAAAAATIAA 6240

TCTGTCT3TT TTTCGTAGAC GCAAATTTTC TAATCTCTTC CAGGIAACG AATCAGAATC 6300

GAAAACTTOG CACATAAAAG TTCTGIGATT CAAAGHAG ATACCCCGAG ACATACACAT 6360

187 851

AGCCGAGAC TCCEAAAGCC TnGTATrTT MACCGGftAA 0GGTK3AADC OSATIACJCSC 6420

TOAAOCCAAT GACATATCCC AACOCUCAC TTCT33CTTT GGTATGACCT GSTACT3TTT 6480

ATTOTTIOST TIGAA3ACTA TTATCCCG TGAIOGTITT GTATACTTAA CCACAAA3CAA 6540

AACCCOTGA. CTIGCATCAC AAflCTTOsAT dTTATCATr CCTOOTSTCA GAAAGTOGAT 6600

GSATACTCCA TT3TTTT3TA AATCACTCCT CKAUGJGACCt AAACTTGΊΊC GA & GTTCGJ: O 6660

TOCTTUACT AΊGΊAGTτTΊ GΊAΊGAAGIΆ ΊC £ CTAAATΆ OaTITCGTC ΊAftTGASATI 6720

AAGTGACA AAOCATGACT CGTSGCCTCT CUGTTGCAZ TCTTTArTZA. GGA3CCT3AA 6700

ΊTΊΊCCGAΊΊ TTTGCGCCC! GAAGATAAGA AAGAAAm ΊGGATCVΊGT CTΊ3A.ΊTΊAΊ 6040

CACO3GAGAA CTOTKATCC ΊGΊCGGTAAΊ AAAGAGAGA. GCCACACAC ΊGAAΊGAGAA 6900

ATGAAAAAT CKEKTLG 'ATATAACAAC ΊΊAΊΌAΊ3AA I3AATΊIGΊΊT ATAMaCCT 6960

TCTTIUTTTA AGGAAAGΊA; Ί CCAAATTTGCC CΊΊΊTTCΊTC GCTATCCCT. AAACAAACA 7020

ATAACCAAA AGAAAAATC ΊTΊCAΊ3AΊΊ AATGUACU GTCTCCCT AAGCCAAAAC 7000

TTTATCTTTA GACCTTTAC CAAATCACA GTATnTAT ΊGCTAGiACΊΊ AGGAAACAC 7140

TlTlTlTllT ACCCAACAAT CΊJΊGGAΊΊΊ UAAUGTOT? TΊΊΊΊCΊACΊ AAAGAUA 7200

CAACTATTA ΊAΊAAΊAATG ΊΊTCΊATC AKnGAOAA? TCnTCTTn 'TAATAAACAT 7260

CCAGCTT3TA raATATCCA. CftAGTCAIT TCACCATTU GGCCATTTA UTTCTTATA. 7320

AAAATAGCA CAAAAAAGAT TATATTOTT TAGCGATT AATTTCTAAT TAACTTACGT 7300

ATT '. ’^ !! TTCCATAGAT ΊTAΊCCTΊCΊ ΊΊTTATJTCC TTAGTTATCT TAGTACTTTC 7440

TTAGTTTCCT TAGTAATTTT AAATmAAG ATAAm GAAATTAAAA GAAGAAAAA 7500

AACTCΊAGTΊ ATACmiTOT TRAATGUTC ATCACACTAA CΊAAΊAATΊΊ TmTAGTTA 7560

ATTACATA TAAAACACT GAAGAAA £ Gr TTGCCCAC ACTTm'HJ3 GATCAATIAG 7620

TATTArT ’ATGGGAAGA.Ί TCTjATTAA AGGATACCAA AAAΊGACIAG TTAGGACATj 7600

AATGAAAACT TAATCTCA ATAACATACA TACGTGTTAC TGAACAATAG TOACATCUTA 7740

CGTGTTTTGT CCATAA '' ’’ GTTGCTTATA AATAATTCA UACAIG ΊΊΊTCAΊTAA. 7000

GCTmAGA. AGCACAAAAC CATATAACAA AAUAAn TCCTATCCCT ACCAAAAAA 7060

AAAATAAAT ATTCCTACAG CCTTSTT3AT TAΊTIΊAΊGC CCTACGTGA GCCΊΊOTΊTA 7920

187 851

CAGTTGCG GTGCICGGGC CATTGCGECI TCGECCAG TCAAOCTCT CGIAATCAGA 7980

ACAAAA333G AAACAAAO3G AAGAGGCAAA ATCCITGGGG GTATGAACTA AGTTGAACGG 8040

CTCTCTSTTT AAGGGGGAGA GGCAAACAEC ATCOCAACTA GAAA3CATTA O3ACTKGTT 8100

GCTTCGTAIC CACCTAAIAT GCCTCIftCTC AATCCTTTCA ATATTTTCA CTTTTTOOCA 8160

OGACTTTC. AAACIGCECT TGAAAACCCA TATCTGTGA ACAGCGGCIG GAGGCGGCGG 8220

AECCAGGA CgAAGAACAC CTGCTTOOC TGCGTAGCTC ACTAACGCTG GGAATAAACC 8280

AACCETIGGA. GTTGEAGA. AAGAOCAAG GGWUIGGGG GGACGAACC GGATCACATI 8340

GTGCTTCAT GATCEICCAG GCAA3AAOOC TCAAGCGTjG ACCGGGGGG AAAGAATTAG 8400

ACCGTCIGTT CTOGTAGAC GCAAATGTGT TAATCTCTTC CACTAAAOG AATCGGAATC 8460

AAAAACTTCG CAOGCAAAA3 TTCTGATT OOGEACTCATA OCA3GCOSAGG TCGAAAGCCr 8520

AAATAETGIA TACCG3AAAG GCTGCAATCC GGTTAEGET AGAOCTAATC ACTTATCACA. 8580

ACTCCTCACI TTTCGGTTTG GTAGGAGCCC ATACIGITET GTTCTTOGTT GOCA3WCEAT 8640

GAGTCCGGT AUGTCTTG TATCATTATA ACAAAGCAAI ACCCCAGGAC GTGCATCACA 8700

AGCETTGATC TTTACCTCIC CGTGTOGCCG AfiAAGOAETC GA3ACTCCTT GGGTATCCAA 8760

AGCGCTCCGC TCAGGAAAA AACTOGTATC AAGTGTGIAT CCTCGTrcJGG AGCTGCTAG 8820

GAAGAECCC. GAGATATGT TOGGTCGAEG GAGATTIAGG TGGACAAAOC AAGACIOGGA 8880

GCGGCCGTTC TGGCACTCGT GAGGGAGCAG OCGCAAGGGG OCGKGGTEG ACCCCCGAAG 8940

ATAAGAAAGA ACCGCTTGGA. TCGTCECCTG AGGGATCACC GGA3AACTC. GGAGCGGATE 9000

GGAAAAAAGA AAA3AAAGGA TGAGCACGAT CAGTCAATCA GADTATAGA AATCAGGATE 9060

GGGAGAGAAC CGACATGAE GAAEATACAA GTCITGAIAA TET.EC3CAAA TTGCCGGGGG 9120

CTTCCTAGT OCCAADCAA GCAAATTAAC CAAAGATAAA AGCGTCATGA AGGTTIGCCG 9188

TTETCTTCGC CAGICCCAGA. GAAAAAGKTA GATAAAAEAT TGCATTAGGT TACGGGTAG 9240

ACCITCAGCC CCAAAGTGCT CTETTGACTI TIAACCAAAI GAACASTAAG TEATAGCCA 9300

GACCTTGA ACAACATGET GGATATATAE GCGTEGACAE CAAAATTCAA CAAECGEGGG 9366

GGGTCGATAG TGIGGGGGGT CGGATGCTAA GAGAGGACCA CGCATGAGAG CAGAGACAAA 9420

GGGGTTCCTG TTCAATCAAC ATCCATTETC TGIAAAAATG AGCAAGTGTC TGCTIATATC 948 ⁸

187 851

TTCTTTCAGC TGTTTTCTTT TTTTATCTTt GGTGTTC3C TITTGCTCC MTTGITTTC 9540

CTTTCTTTGT TTTTTCTTT TATTTTTCTI MTTT3TTT TITTTTAATT ATTTEITTT 9600 tctcctgttt atctoctgtt attkotict ttctctctt tsgtctttt cttttttttt 9660

TTTTTGCTTI TGTAAAAT ATTTCTATrC ATCTTTGT TTTTGTTGGC TTTTTCTCTT 9720

ITTTTTITTG GCCAATTTG? ATTOGTCTT TTTGTTTCT TTUGTTTTC CCTCTAITTC 9780

GGCGAAACGT TTTTTCGTT? TCTTAAGTIA GGTTTTTKTG TTTTTGTGA 7TGGGTTTTC 9S40

TTTTCTCTCT CCTGAAT3T GTATGITGA AACCCCTTTT TTTTTTGGTC (GTTTCTTGA 9900

TCAAGATGIT CTCTCGTI 9919 (2) INFORMATION FOR SEQ. ID NO: 2:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 5655 base pairs (b) TYPE: Nucleic acid (C) STRAND: Single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTISENSIVE: NO ( ix) PROPERTIES:

(A) NAME / KEY: exon (B) ITEM: 2787..3347 (A) OTHER INFORMATION: / product = 1st exon with NIM1 (ix) PROPERTIES:

(A) NAME / KEY: exon (B) ITEM: 3471..4162 (A) OTHER INFORMATION: / product = 2nd exon with NIM1 (ix) PROPERTIES:

(A) NAME / KEY: exon (B) ITEM: 7271..7777 (A) OTHER INFORMATION: / product = 3rd exon with NIM1

187 851 (ix) PROPERTIES:

(A) NAME / KEY: exon (B) ITEM: 4586..4866 (A) OTHER INFORMATION: / product = 4th exon with NIM1 (ix) PROPERTIES:

(A) NAME / KEY: CDS (B) ITEM: joh (2787..OT, 3427..411624271 ... 4474.4586 ... 4866) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

TCTSTTKGA GTC3TTOO3TI TTTGCTTTTCT GTTTAGTTTT CTTTTOCTTC TCGTK3GATTC 60

TTKGTCTTCA TTCCTTOCTC TTTTCTGTTT CTGGTCTTTC CTTTGCfflGA TGTGTT03GT 120

TGGGTTTTTCT OTT33GTTT TGCGGTTTTC GGTTTGTTOC CTTTCO3GTT TTAAATTCAT 180

TGGCTTTCGC TGTCTCGGCG TTGTGTTTG TCTO0GSGTT TCTTCCTn GTTTCTCTGA 240

TCTTTTTTCT GCGTTGTTTT OGTTTCTGTT TTGTTTTCCT GGTTGTGT TGTTATTTTG 300

CGCTTCCTA TTTCTGTCTG TTTTTTTTTT TCCTTCCGGA TTCTTGTTTC GGGGTTCTTG 360

CTTTGGTTTT CTCGGTTCTT CTTTGTGTTC OGGTTTTGTC TCATTTCCGT TTCTTGGTCC 420

TTCTTOCTTA CTCCGTACTC TGTTGTTTTO TfflGGTTTTG TCTGTTTCGT TGGGTTGCTA 480

GTK5OTTTO GTCTGTGGTC TTTOTTTGTT CTTGTAGTTG TTCTOGTGTT TT3GGTTTTT 540

TTGTGCTGTT TlfflTTTGIC GTGGGTTTTT GTKATTflT TTTTTTGOTT TGGTGTTGTT 600

TTGTTTTCGT GfflCTOCTTG CTTCfflOGTT GTTTTGTTTC GTTGTTGTGT TCGTGGTT3C 660

CTCTTCTTCA TTTTG3OT3T GTTGTTGTTT TTAGTTTTIG OTTTTTTTGA GGTTCTGTT 720

TCTTCTTTOG TTTGTTTTCC GTTTTGTTOT GTTITCGTGT G9GT®TOT fflTTOGTTGA 780

TGCTTOGGGC OGTOTCTTTT TTTTTTTTTTT TTTTATTATG GGCOGTOTTT TGCATTOGTT 840

GTTGTOG TTOTCTTGTC TCTTGTCTCT TTTGGCTCGC TCTTTGTGGG GCTTTTTTTT 900

TTCTT3K3TT GTTTTTTTGT TTTTTTTTTG GTTTTTTGGT TTTTTGTTTT TTGTTTCTTT 960

GGTTTTTGTT TTTTTTOCTT TCTTTGTTTT TTGTTTffiTTT ATTCTGTTTT TTTGGOTTGT 1020

TTTTTTTTTT TTTCATGGGT TCOTGTTTTT TGTTGTTTTO TGTTTOT3TT TTGTTTTTTG 1080

187 851

TCTCOtCAAT Imn .CTTCTIAIA TAGTIAIIA TTTITTTI AADATA1AG 1140

TITTTOITC TAICΊACT3O TTOTATTT IA3COCOCCO COS: nI®IO TTACATAGT 1200

ITCOACICI ITCICGOIIA OoTAITTCA OO3ICTOTC GITIIIIAAG OTi ^, TCITOA 1260

CIAΊATGICC GGICT ^ OIGI COAOIIATC CIATTOIOOC CCCCCCCO ^ O CTQG1TΏOC 1320

TTAITOITT CCTA3iTIGGO CCTIGGC3I3Γ TTAOTCCCG TΪTAΓOCOCC ITΠT'TCT 1380

OIA. ^Γ CCICGC T ^ GIA3CCAC ICTCCCTTT CICICCGAIT GATIOCAIIC AATTTTATAA 1440

ICIATOCTT ^, ICCAAATΊA CTICACA3CA CCOICTΓTOC ICCACTT3Π 'OCCCA3CITA 1500

TTCJICCCAI ^l ICATTCATC T3IGAIGOIC TTGAGTTATA GASAGTTII GΊICAflΊTCO 1560

ITCCCCTOCI COCCAIOΓEC TCCTAATITA ACTIAICArr ΊTCGCCTTO CCCCGICACC 1620 ^ 0203 © ^ GOACICCΠT ATTTACCTTA ^l ITCIACOICO IAIAICCCGΓ ACICIGITA 1680

ITOCACCICC TOT [AO; 3ITC TIGTCΠOCI CGGCCTO ^, 3T 'CCCOIA] TOT TIOAΓΠTOI 1740

GCTOIO3CAO GI · !!!!) © !: CAACTCTTG CGIOTAOCG3 TITITGTT 1800

CIGCA3AIIO CΠOITOΓTO ICAGTTOOC GOCCOCAO3C GIOO ^ G & CCA OACCTTOCC 1860

GTGAAGGAG CGOCCCCC ^, ΓT GITΊIC3CCI GIICIGACΠ 'IGOΠ ^, ΓCOG TOGTAGTTAT 1920

IGCCACA3OΓ GAΠΊATO ^ O TICCTICAGC ACGGAIT GCCG ^ OACA? CCOICCCGCC 1980

GIOTTATA ToITTTOTI CTΓIITm ^, Γ TTIOCTTC CTTCIITTT CTAIITCTΓO 2040

ATTAIGACIT TCATGTTfrT AATIlTAnT T3TOOITTCΓ OIAICTTICI ΊΊTGITIIIT 2100

AAICirrTI CAITIGCIAI TITACCGIT ICTI0ITIOO TGGTTITT C3COTTTT 2160

TAATAKGT GITCTCCGO TIACATOIIC CTC.TTACΊT TTOTATCAC IATTACIAT. 2220

TTCIOCCIΈC CTCTTΠTA GITRTTCTI CTIITTCII TCIAICOGΓΓ OOCTΓTKΓCΆ. 2280

TTIACTICA CCGCACATTC TOAOATTIΓ CTOTt ^, IOACC IσITCCIOIC TΓTOTTTC ^, 2340

TICTATTIC ATATTTATAT ATTTKTKTGA CCTICAOIAC OOIIITICCC CTIOTTTΊT 2400

ATATATTTAT TIOAIOTOCC CT: IOT: ϊΠT HTTCAGIII CITCCOGTT: CI © CITGAA 2460

IIOIoCCIAT CCCATCΐTI CTTToτAC TIIOIOO3IΓ AIATTACΪA COCIIΊCICT 2520

COOOITTCI ΊT ^, ICGCΠCO IGIIATATT TTTICCCCAC ITΓOTO [GCO ACCGCTTOOI 2580

TICOTI3ATC TTTACCGGIT TTI3'T3CAΓ GACCOOGII GGACGAGGAT IOITOITCAI 2640

187 851

ATCITCCOAC CACIOICGTr GOTTGAOT GGOCTGCC GTOATUT ATTCTTCATC 2700

TITAACCAAA TOCSGnGAr ΑΑ3ΏΤΊϋΓΓ CGnGATAG CA3AGACTC TTAATnGT 2760

GATTKCaA TCATC3GAAC CTGITG ATG GACACCACC ATT GAT GGATTC GOC 2813 Mt Asp Thr Tir Ile Asp Gly Phe ALa

5

SAlTOTTKTGAAATCAGCAGCACTAGfTT ^ CTC GGTACCSAAAAAAC 21861

Asp Ser Tyr Gu Ile Str Ser Tir & t Phe Val Ma Tir Ap Asn Tr 10 15 20 25

GA TCC TCT ATT GTT TAT CTG GOC GOC (GAA CA GIA CTC ACC GGA CCT 2909

Ap Che Ser Ile VsA Tyr Leu ALa M.a Glu Gln Val Lu Tr Gly Pro

35 44

GAT GTA TCT GCT CIG CAATIG CTC TCC AC GC TTC GAA TTC (GT ITT 2957

Ap VlL Ser Ala Leu Gln Leu Sr An Sr Hej Gu & f Vv1 Phr

50 55

GATTΌlTCGGAΓGAIT'TTTATAGCClA (GC ^, M3CΓT GIT CTC 1TC (GC 3005 An? Ser ho An? An? Rre Tyr Ser Ap AAa Il® Ilu Vv1 Ltu & r Anp

65 70

HCCUG ^ GIT TCT TTC CAC Π TOC (GT TIG TCA GCG AA AAG ICC 3053

Gy Arg Gu VaL Sto Pre Hs Arg O Vvl ILu Sf Au A See See

80 85

TTC ΊTTAAlAIC GT TTA lCTGTCQTTAAβ AAG GAGAA (GA TTC AAA 3101 hre hre Lys Ser ALa Leu Ala Ala AAa iLy L Gu ILy A SSe An

95 100 105

AACAC: GCC GCOGG AAGCIO OT AG (GA ATT CGC MG GAA TTA 3149

Asn To ALa ALa VML Lys Leu Glu Ilu Lls Glu Ile AAa Lls Aap ITs

110 115 112

GAAGC GG TTCGA TCGGT GIG AAC GT TTT GCT TAT (GT TTA CAG 31.97

Gu VćA Gy ETe Ap Sto Val Val Tr Vv1 Ilu AAa Tyr Vv1 ITs Ssu

125 110 115

AlCAGAGTIAGS CCGCCG CCΓAAS GG OTT ΊΓΓ GAATG (GCA GGA GGA 3245 Ser Aog VaL Atoj Eho Ppo ho Lls Gy Vv1 & e Gu (Csi AAa AAp <Gu

140 15 (0

ASS'TΌCTCTCSCGTGGTTTG; TGG ¢ TCIClOI (GG (GA'TI'T GAT HG GGA 3293 An QS3 Cys łtis Vv1 AAi (Ob AA) ho AAa Vv1 AAp Pte ffet Ilu GG

155 160 116

187 851

GA CTC GA * GC GA ATC ATC TTT AA ATC CCT GAA IGA MI / CCC CTC

Val Leu Tyr leu Ma Pir ile itoe Lys Ile Ras Gu Lu Ile Thr Leu

170 175 180 1185

TAI CAG GAAAACACC ATCTGCAC AGCCGAGGrT ACACATTCAT (GAAIAIG Tyr G-n

TTACTTaGT TACCGIGIAG (GMTAGIG CAG GG CAC AGA (TG {AT GI TA?

Arg H.s Leu Leu Ap> VaL Val / Ap

190 199

AAA GII GIT AIA GAGGACftACTGiGEITAICCCCAGCGGIC AAA TAI Lys Val VaL Ile Gu Ap Ihr Leu VaL Ile Leu Lys Ilu. He has how many

200 205 210

TCT Gd AA GCT TGA AAG CG CTA ATC GAI AGA “GA AA (SA ACG jAT

Cy Gy Lys ALa Cy Mt Lulu Lai L ^> paj CCsI-L ³ d He Ile

215 222 225

GAC AAG TCT AAI GAA GAI AG GA AGT CAT <AACAG ΊΑ HA CCG GAC Vl Lys Ser An VH Ap Met Val Ser Lai Glu Le Tee Ilu £ Po Glu

230 223 220

GG CAG GAG AAC GAG AGA JGTT GAI AGA CGEG JAA (AG (CA * CU TAG GAG Glu Lee VlL Lys Gu Ile AiasAdj Aja Lu GGlIlu Gy iee Gu

245 250 2855

GGA CCG AA GTAAA3 CA CA GGCT03 CAG GA CAT AGCA CAT GAC Vl Pro Lys vaL Lys Lys Hs VaL Ser Asi Val me Ly Ala Leu Asp 260 265 270 225

OG GAT GAG AEI GAG TA GCT AG TG CAG GIG AAC (GA © ACCA / AC

Ser Asp Ap Ile Gu Leu W. DyiLn Lei Ul tyB & u JAp JHa Tir

280 285 229

AG CIA GAI GAT UGIGA GCI CII AA AC GCT GA CGC AIA1GG iAA

An Leu AP Ap ALa Cys Ala Lei His Re »Ala Val Ma Gir CCs / Asi

295 330 335

GIG AAG ACC GCA ACA GA CA (IA AAA CII Cd CA GCC CGA (GI / AA

VI Lys Ar ALa Ar Asp Lu Leu Lys iuu Asp 1 ^ AAi Αρ VVL Aasi

310 331 332

COI CGG CCI CHCG GGC AG AG GG CII CAG GU GCC GCC GAT CCG

His Ag Asn Rro Arg Gy 1 \ i In VM Leu Lis W / Aa Ma JMt tar

325 330 333

AAG GAG CCC CCC GTG ATC CIC AGC IC AGI GG. AAC CGG JGC CCG JGC

Uy3 Glu Pro Gln. Lee Ilu Leu ue · Lai Leu ulu Ly? Gy Ma See Ma

340 345 350 335

3341

3397

3450

3498

3546

3594

3642

3690

3738

378G

3884

3882

3930

187 851

TCA GAA GCA ACT TIG GAA 03Γ AGA ACC GCA CTC MG ATC GCA AAA CAA 3978

Cheese Glu Ala Thr leu Glu Gly Arg Bir Ala Leu Met Ile Ala lys Gln

360 365 370

GCC ACT ATG GCG GIT GAA TGT AAT AAT ATC COG GAG CAA TGC AAG CAT 4026

Ala Bir Met Ala Val Glu CyB Asn Asn Ile Ero Glu Gln Cys Lys His

375 380 385

TCT CIC AAA GGC OGA CIA TUT CJIA GAA MA CIA GAG CAA GAA GAC AAA 4074

Cheese Leu Lys Gly Arg Leu CyB Val Glu Ile Leu Glu Gln GHu Asp Lys

390 395 400

OGA GAA CAA ATT OCT AGA GAT GIT CCT COC TCT ITT GCA GIG GCG GCC 4122

Arg Glu Gln Ile Pro Arg Rsp Val Pro Pro Ser Phe Ala Val Ala Ala

405 410 415

GAT ®A TIG AAG ATG ACG CIG CTC ®T CIT GAA AAT AGA G 4162

Asp Glu Leu Lys Met Thr leu Leu Asp Leu Glu Asn Arg 420 425 430

GIATCIATCA AGILT1A1TT CTIAIMGIT TCAAITAAAT TTATSTCCTC TdAITAGGA 4222

AACIGAGIGA ACEAATCATA ACIMTOTT GIGTCGIOCA CIGITIAG TT GCA OT 4278 Val Ala Leu

435

GCT CAA CGT OT TTT CCA AGG GAA GCA CAA GCT GCA MG GAG MC GCC 4326

Ala Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile Ala

440 445 450

GAA ATG AAG GGA ACA TGT GAG TTC ATA GIG ACT AGC CTC ®3 CCT GAC 4374

Glu Met Lys Gly Thr Cys Glu Ehe Ile Val Thr Ser Leu Glu Pro Asp

455 460 465

OT OT ACT GGT AO3 AAG ACA ACA TCA COG GGT GIA AAG ΑΙΆ GCA CCT 4422

Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys Ile Ala Pro

470 475 480

TIC AGA ATC CIA GAA GAG CAT CAA AGT AGA CIA AAA GCG OT TCT AAA 4470

Ehe Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys

485 490 495

ACC G GDOGGKITC TCACCCAOT CMCGGACTC OTAICACAA AAAACAAAAC 4524

Thr

500

TZAATC & TCT TTAAACAIGG TTTTGITACT TGCIGIOTA ΟΤΤΰΠΊΊΤ ΤΠΑΚΑΚΑ 4584

187 851

G TC GSA CC (GrAA (GA. TIC T1C CCG CJCC TCG (CCA GIG <UC 4629

VaL Glu Leu Gly Lys Acg Phe Phe Pro Arg Cys Ser Aa V <OL Leu

505 510 515

GAC CACAH 'ATG MA CG ®G GAC TTC TAT (CA (CT Gd TTCGGA (GA 4677

Asp Gn Ile Mat Am Qc Gu JAp Ilu Ur Gn Ilu Aa «Cs Gly Gu

520 525 530

GAC GAC ACT GC ^ GWC AAA CGA CTA CCA IAA / AC CCA ^ Χί lACATG GA 4725

Asp Asp Thr A.a Gu Lys / Ag Ilu Gn Lys Lys Gn / Ag Title: Gu

535 540 545

ATA CAA GG ACA CC1AAGUZAQUCTΊTΊCUGAGUA / Cff UG GAA HT 4473 Ile Gln Gu Thr Lai L / jl lys Aa fPr Sfer Gu Ap An Hu Gu Ilu

550 555 560 <GG AAT ΊCU TCC CCC GA AGT TTG / AC TCC UC CAC TCG AAATCA AAC 4401

Gy Asn Ser Ser Lel Lt / Ap Seu Thr Ser Ser Thr Se Lys Ser Ur

565 570 575

GT GAAAGAG> ICI AAC CG AAA CTC ΊCTCTΊCCΊ OG ¹ O3G TG 4406

Gly Gly yys Arg Ser A3n Ag Lys Lau Ser His Arg Ag Arg *

500 505 590

GACICITCOC TCTTACTCIA ATTITIGCIG TACOADAIAA nCIGTITTC ATGATGACIG 4926

TAACTCTrrn. Ί ^ αΑΚΕΤ TGGCGICATA TAGTTTC3CT CnCGmIG CATCCT3TGT 4906 ^ IAWCIG CAGTTCTCCT ICAAACAAAT GTTGTAACAA TTTGA00AA IGCEmCAG 5046

ATTTGTAATA TATATTCiTC I3C3ICOACA AIAACCCATC ATGGTGTTAC AGAGnGCIA 5106

GMO ^ IAGT GTGAAATAAT GTCAAATTCT TCATCIGTTG GATATmCC ACCAAGAACC 5166

..AAC ^ IA! TCAAGnOCC IGA ^ ITC ^ GCACATOCA TGTTATATGT ATCITCCTAA 5226

TTCTTCCTrr AAOCrrnGT AACICGAftII ACACAGCAAG TO.3mCAG GrCIAGAGAT 5206

AAGAGAACAC IGA3IGGGCG IGIAKCGIGC ATTCTCCTAG ICAGCICCAT TGCKTCCAAC 5346

ATCIGIG ^ GACACAAGTT AACAAΊKCTT IGCACOOT ^ CTGCGΊGCAT ACAIGGAAAC 5406

TTCTTαGATT GAAACnCGC ACAIGIGCAG GTCCCTT0CC TGTCACTCAT AGACCAAGAG 5466

ACTcAAAGCT UCACA ^ GCCCTCAAAT C1TCTCTTTC TAT0GTCATC ACTCCATATC 5526

ICCGACCACI GGCAUCAGC CAGAGCCCAC IG / HIUGAG GGARnOGGC TAACCATTTC 5506

CUACCTTCTG AGTCCTTCTT TTTGAIGTCC TTTAGIAGG AAICAAAIC ^ CCTCCIGA 5646

CΊTGTCGAT

5665

187 851 (2) INFORMATION FOR SEQ. ID NO: 3:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 594 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Mt Aup Thr Thr How much Aup Gy He Gla aa Cheese II? Gu How much Cheese ^ r 1 5 10 15 Thr Cheese Ehe VVL / Aa Hr Ap Aun Ir Aup Cheese Cheese How much ul Tyr Leu twenty 25 thirty ALa Ali Gli Main ul Lei Thr Gly Pro Aup Vd Cheese Ali Lei Gn Lei 35 40 45 Lei Cheese Aun feer Phh Gu Cheese Vd PłP AiA> Cheese Prr Ap Ap PAu The 50 55 60 Cheese Aup Ali Ljl UeL VæV Llg Cheese Ag? Gy Ag Gi UL Cheese Ple IHs 05 70 775 80 Arg Ceu vh Lei Cheese ALi Arg Cheese Cheese PiPi Phe LiL uer Ala How many / Aa 85 90 95 ALi Ali Lys ILS Gu Lls Aup Cheese .Aun Aui Tr Ala Alu Vd Ll? How many 100 105 110 Gi Lei Lys Gu How much / Aa Lys soups up / y Gu llal Gy irne Αρ & rr Vd 115 120 125 Val Thr ul How many 7Aa Yttrium Vd iyr Cheese Sre A3 Vd Ai3 Pao Pro Pro 130 135 114 Lyu Gy Wow Cheese Gu iys / da Au? Gu Aun (Cr Cys H.u Vd Ali thousand 145 150 155 160 Arg Pro Ali ld Aup Phe tet LeL Hg Vd LU Τα L ^ i Il Pte How much 105 171 175 Phe Lyu How much EPo Glu How many How much Thr ILei T ^ h Main Arg His Lu How many Ap 180 185 190

leu Llg Leu

Val ld Aup Lyy Val Val Ile Glu Aa? H Leu Vd Ile 195 200 205

187 851

TLa Tsn He Cys Gy Lys TLa ^ ys Mst Lys Leu Leu Tsp Tog Qys Lys 210 215 220 Glu Ile He VæłL Lys Cheese Tn VOL Tp Underworld vh Cheese Leu Gu Lys Cheese 225 230 235 240 Leu Pro Gu Gu Leu VaL Lys Gu He He Asp Tog Tog Ly Gu Leu 245 250 255 Gly Leu Gu W. Pro Lys Val Lys lys Hs Forest. Cheese Asn Val His Lys 260 265 270 TLa Leu Asp SIot Asp Tsp. How many Glu Lai VaL Lys Leu Leu Leu Lys Glu 275 2S0 285 Tsp Hs Thr LTn Hu Tsp Asp Ta CC® Yeah Leu Hs Phe Yeah VaL Tli

290 295 300

Tyr Qys Tsn Val Lys Thr Tla Thr Tsp Leu Leu Lys Leu Tp Leu Tla

305 310 315 320

Asp vaL Asn Hs Arg Tsn Pro Arg Gy Tyr Thr V «aL Leu Hs VaL TLa

325 330 335

Tla Mit Arg Lys Glu Pto GLn Leu Ile Leu Ser Leu Leu Glu Lys Gly 340 345 330

ALa Ser Ala Ser Glu Ala Thr Leu Glu Gy Arg Thr ALa Leu Mit He 355 360 365

ALa Lys Gn TLa Thr Mit Ala Val Gu Tsn Asn He Pro Glu Gln 370 375 380

Os Lys Hs Ser Leu Lys Gy Trg Leu Qys VtOL Gu He Leu Gu Gln

385 390 395 400

Gu Tsp Ly Tg Glu Gln He Pro Arg Tsp vaL Pro Pro Ser Phe TLa

405 410 415

vil TLa ALa Gu Leu Ly Underworld 420 Val TLi Leu Tla GLn Trg Lsu Rh! 435 * 10 Gu How much Tla Glu IMt Ly Gy Tho 450 455 Gu Pro Asp Tog Leu Thr Gy Thr 465 470

TLy Iee Ter Asp Leu Glu Api Leu 425 433

LPr> Thr Gu Tla Main Tla Ala Mt 445 Cys Glu Phe He val Tir Cheese Leu 460 Lys Arg Thr Cheese Pro Gy vh Lys 475 480

187 851

Ilt Ala Poo hhe Arg Ilt Ltu Gu G.u H.n Gln Sto Arg Leu Lys A.a 485 490 495

Leu Sur Lys TIo Val Gu Lu Gy Llss Aog Pre Pirs Poo Aog Oss Sto 500 505 510

ALa Val Leu Asp Gln Ilt Mt Asn Qys Glu Asp Lu Iro Gn Leu A_a 515 520 525

Cys Gy Gu Asp TA? Tho ALa Gu Lya Aog Lu Gln Ls LS Gn Aiog 530 535 540

Tyr Mt Gu Ilt Gln Gu Ter Lu Lys Ly Ala Pee Seo Gu Asp Asn

545 550 000 560

Leu Gu Leu Gy Asn Seo Lo Leu Tur Asp Seo Tho Ser Seo Tro Sto

565 570 575

Lys Lr Ter Gy Gy Lys Aog Sr Asn Aog Lys Lu Lr Hs Arg Arg 580 585 590

Aog * (2) INFORMATION FOR SEQ. ID NO: 4:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 amino acids (b) TYPE; amino acid (c) STRAND: not applicable (D) TOPOLOGY: not applicable (ii) MOLECULE TYPE: protein (x) SEQUENCE DESCRIPTION: SEQNO 4:

Ilu Arg Aog Mt Arg tog Li Asp MLa Ma Αρ Ile Gu Ilu VG 15 10 15

Lys Ltu Mt VM Mt Gly Gu Gly Lu Asp Li Ap Asp ALa Lu Ala 20 25 30

Val His Ty Ala Val Gln HLs Cys Asn

40 (2) INFORMATION FOR SEQ. ID NO: 5:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 amino acids

187 851 (B) TYPE: amino acid (C) STRAND: not applicable (D) TOPOLOGY: not applicable (ii) TYPE OF MOLECULE: protein (x) OPS SEQUENCE: SEQ NO: 5:

Pro Ihr Gly Lys Ter ALa Leu Hs Lu d.a da GIu MmI VæOL Ser Pro 15 10 15

Ap Met VaL Ser VłL Leu Lsu A? Hs H.s da A? JXa An PPie Ag 20 25 30

Tr Xaa Ap © y VaL Ter 35 (2) INFORMATION FOR SEQ. ID NO: 6:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 amino acids (b) TYPE: amino acid (C) STRAND: not applicable (D) TOPOLOGY: not applicable (ii) Molecule TYPE: protein (X) SEQUENCE DESCRIPTION: SEQ NO: 6 :

How Much Arg Arg Ftet Ag .Arg Aa Leu A? will give A? How many Glu Lei Vd 15 10 15

Lys Leu Mt VVL NMt Gly © u © y Lei Asp Leu Asp A? da Lu da 20 25 30

V = d. H.S Tyr da Val Gln Hs Cs Asn 35 40 (2) INFORMATION FOR SEQ. ID NO: 7:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 amino acids (b) TYPE: amino acid (C) STRAND: not applicable (d) TOPOLOGY: not applicable (ii) TYPE OF MOLECULE: protein

187 851 (xi) SEQUENCE DESCRIPTION: SEQ NO 7:

Arg Arg Pro Asp Ser Lys Thr Ala Leu ffi.s Leu Ala ALa Gu Mt VaL 15 10 15

Ser Pro Asp MU VaL Cheese VælL Leu Leu Asp Gln 20 25 (2) INFORMATION FOR SEQ. ID NO: 8:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 amino acids (b) TYPE: amino acid (c) STRAND: not applicable (D) TOPOLOGY: not applicable (ii) MOLECULE TYPE: protein (X) SEQUENCE DESCRIPTION: SEQ NO: 8 :

Ile Arg Arg Mt Arg Arg ALa Leu Asp ALa Ala Asp Ile Glu Leu Val 15 10 15

Lys Leu Mt VłL Mt Gy Glu Gly Leu Asp Leu Asp Asp A.a forest TAa 20 25 30

VaL H.s Tyr ALa VaL GLn H.s Cys Asn 35 40 (2) INFORMATION FOR SEQ. ID NO: 9:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 amino acids (b) TYPE: amino acid (C) STRAND: not applicable (D) TOPOLOGY: not applicable (ii) TYPE OF MOLECULE: protein (X) SEQUENCE DESCRIPTION: SEQNO: 9:

Arg Arg Pro Asp Ser Lys Thr Ala Ilu HHj Lsu Ala Ala Glu Met Val 15 10 15

Pro Asp Mt VgL Cheese VaL Leu Leu Asp GL.n 20 25

187 851 (2) INFORMATION FOR SEQ. ID NO: 10:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 amino acids (b) TYPE: amino acid (C) STRAND: not applicable (D) TOPOLOGY: not applicable (ii) TYPE OF MOLECULE: protein (x) SEQUENCE DESCRIPTION: SEQ NO: 10 :

Ile Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile Glu Leu Val 15 10 15

Lys Leu Met Val Met Gly Glu Glu Lep Lep Aeu Asp Asp Asp Ala Alu Ala 20 25 30

Val His Tyr Ala Val Gln His Cys Asn 35 40 (2) INFORMATION FOR SEQ. ID NO: 11:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids (b) TYPE: amino acid (C) STRAND: not applicable (d) TOPOLOGY: not applicable (ii) TYPE OF MOLECULE: protein (x) SEQUENCE DESCRIPTION: SEQ NO: 11 :

Pro Thr Gly Lys Thr Ala Leu His Leu Ala Ala Glu Met Val Ser Pro 15 10 15

Asp Met Val

187 851

Γ4 ώ

187 851

Fig. 3

Φ <α ε »

c.

Imr>% ο

♦

ο.

ο

187 851 ca g

what aa

CD in aa ua

CD * C

CD

5 ±

WHAT '

CM

Of the Jagiellonian University

ABOUT

And β

«A

• them

And u

fc.

pp

CM

CM

CO r * CM

ABOUT

TT ca <£>

A * fl Sn

N * .§ aE _O r) 2 • 'Β'& δ

SPnSJs fea ^ S

187 851

U83.1 -4- U84.1 -4- L84 -14-- U84.2 -17-UL84.4 & 5

44 40 26

CIC12H07

t. ' ^{* 1} .:' --ί

CIC12F04

1 2 3 4 5 6 7 l ' ¹ I' 1 'II' I '''i 1 I 1 II 1 III ι 1 IIIII ł l I l i-

CJC7E03

Fig. 5. Schematic representation of the YACs flanking Nim against the genetic map and AFLP maokeos. The physical distance in nx100 kb is shown below. Only marker positions W84.2 __________________ ___________ ₁₁ ych Nim I idem the genetic map and aFlP maokeos. honlower. physical volume in nx100 kb. Only the positions maok ^e 0 and W84.2 were accurately labeled, the remaining markers can only be specified up to a certain range.

187 851

W84.2

Ρ1-2

1-1

CIC12K07

C1C12F04 :::::: - - ... —i

About 100

L-J - I L 1 I 1 I I! I i i i

200

1111 I I l I l

300

400

J-l a-i,

W85.1 U8Ó.1

P1-3 i i i '' i i i i i i t i i l i 1 i

-L-t- 1 1 I 1.1 t-1 ..... 1-1, 1-L, i

FlS. 6. Consumption of P1 and BAC clones for flanking AfLp and YAC markers. The positions of the P1-3 and P1-4 clones relative to the YACs 10G07 and 7E01 have not been determined. The physical value in kb is shown in doLe. Physical distance and tab ranges are best estimates and are not exact values.

187 851

W84.2

Ρ1-2

I-1

Pl-1 RAC-01

I-1 | P1-6 P1-7

I-1 ICIC12H0?

CIC12F04

O 100 200 300 400 J I I I I I J I. J I l l-J.J. I 1. I I I I I I I I I I I 1 1 I I I I I I I I I I Γ i

U85.1 W86.1

P1-3

AND-

P1-4

P1-9 t t I I I J 1 1 1 ł I 1 I I f »r I I I I» ł I I t * ł I t I I I I I t I Ł

X ,, xxl

J_X

Fig. 7. Extended P1 and BAC contig covering the south end of YAC CIC12F04 and flanking markers. Clones Pl-3, Pl-4 and Pl-9 completely overlapped with Yacami 10G07 and 7E03 and were not physically seated against these YACs. The physical distance in kb is shown at the bottom. Physical distance and tab ranges are a best estimate but are not exact values.

187 851

W84.2

Ρ1-2 BAC-03

I-J IP1-1 BAC-01 BAC-02 | -1 1-1 (-1

P1-6 P1-7 P1-16 | -1 | -j | CIC12H07

P1-18

P1-17 LP1-21

CIC12FO4

About 100 200 300

I 1 ι I t i I> 1 ι I ι I t I I I 1 I r I 1 1 ł I t I 1 1 ι I400

I I I .1

BAC-04 / F1-20

-j

P1-Z3 / P1-24

- | | P1-2Z

And BAC-05

WS5.1 U86.1

PI-3

P1-4 i

IP1-9

BAC-06

AND-

500

1.1,, t I ..! ..,!

600 i i i i 1 i i i i 1 ....... - I I i ί I l l I l l l l l 'I ’

Figure 8. Schematic representation of all identified P1 and BAC clones and their relative positions in. area of Him. The physical distance in kb is shown at the bottom. Physical distance and tab ranges are the best estimates but are not exact values.

187 851

Pl-17

6a

6b

Pl-21

Pl-18

BAC-04

40 60 80 100 i i l l I I i i l -i 1 l l l 1 i I ι ι i I -I l L 1

9b 9a 9c

Pl-23/24

BAC-05

Pl-22

BAC-06

120 140 160 180 200 kb

J — 1—1 — I — i — I — i — I — I -! -! I 1 I Ll I l l.l i l.l i I l l-l

Figure 9. Integrated detailed genetic and physical map of the NIM area. The scale at the bottom is in kb.

187 851

P1-18

L84.6a L84.6b

BAC-04

L84.7

A4 (15)

C2 (22)

A10 (22)

Ali (20) ł ^- (18) | 83 (21)

A8 (19)

C7 (22)

10 20 I i i 'i l t j i i i

SgrAI

L84.C

40 50 60 70

I I I I j l I ... L I | i I I l I l ł I I I l ι ι ι I

SgrAI SgrAI BssHll

L84.8

-......... 1 | BAC-06 j _1 1_____ 1 ! os (21) -...... | E8 (22) I and 1 E7 (22) | 1 t 07 (22) —-1 I- E1 (21) 11 1 J 1 E6 (23) 1 t - ¹ ' :-and 1 1 F12 (18) 1 ..... , ¹ 1 1 1 80 90 100 1 1 I 1 110 120 "and 130 140 150 1 1 ι 1 1 1 i t 1 1 1 J. I 1 * I 1 1 1 I I 1 t i 1 i * 1 1 1 I 1 1

SgrAI SgrAI BssHll

Fig. 10. Integrated map of the NIM area. The map is the best match of AFLP fingerprint data and restriction mapping. Relevant AFLP markers are shown. BAC and P1 clones, cosmid contig, cosmid insert size (in parentheses) and some restriction sites. The scale at the bottom is in kb.

187 851

L.84.63 L84.6b

L84.Y1 US4.Y2 LS4.7 L84.6c

1 1 I l AND 'BAC-04 | | 1 1 1 •and P1-1B 1 1 1 1 and 1 and 1 1 AND t 1 1 | j A4 (15) 1 11 | C2 (22) , ¹ and ¹ . 1, II A10 <22> 1 -, - II- I A11 (20) --1 | '02 (18) I _

| B3 (21)

A8 (19) | ] C7 (22)

20 30 40 50 60 70 80

t 1 1 1 1 1 1 1 t 1 1 t 1 <1 1 t 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 | 1 1! 1 1 1 1 1 ₁

SgrAI

SgrAI SgrAI 8s $ HII

L84.Y3 W84.Y4

L84.T1 L84.T2 184.8

BAC-06 [|

L · ’-.....

| O5 (21) | E8 (22) (22) | E1 (21) |

F12 (fc ł H— ~ E6 (23)

E7 (22)

100 110 120 130 140 150 160

I ..... 1 ... 1.1 t.l, .1 ,,. 1.-1

J- i i i 1 i i i i I i i i i I i i i i l i, i i j I ι ι i

SgrAI SgrAI BssHII

Fig. 11. Integrators mipi obuziri NIM include the new AFLP mirkers. He mimics just a little dope from the fingerprints of the AFLP pilots and the reutrician ones. The pokizine will include relevant AFLP mirkers, BAC and P1 clones, the koumid contig, cosmid tip sizes (in the lowlands), and some restrictive sites. Skili and the bottom are jeut in kb.

187 851

L84.6a L84.6b

L84.Y1 W84.Y2 184.7 L84.6c L84.Y3 W84.Y4

0169:

Ler

Us

C105:

10 20 30 40 50 60 70 80 90 i1 · 1 1 1 1 I 1 I t 1 III III II 1 1 IIIII and IIIIIII l II l II | II _t 1 j II

L84.T1 L84.T2 L84.8 l84.9a L84.9b L84.9c

Us

Ler

100 110 120 130 140 150 160 170 180 lii I I I 1 1 I I I 1-1 J-L-Ł-l-l i i i I i i i i i ...... 1 ..... 1, 1 f 1 I I I 1 I I I I I i I I

Fig. 12. Schematic representation of the major Dlo9 and Ci05 recombinant genotypes. A recombinant chromosome is shown with the Ler part indicated as a black bar and the WS part indicated as a gray bar (the non-recombinant chromosome is of the Ws type). The top shows the positions and approximate distances of the respective AFLP markers relative to the physical size in kb as shown below.

187 851

187 851

these

187 851 t »

g &

Fig _e 13c

187 851

XI

Ό to • I · * te

187 851

Length: tgatcatgaa agaaggcggt caagagattg caattggtcg taactaagtg cttcaacttg aacccactct caaagctgcg gtattgccta tggactgcaa gcgcgttttg cctctgatca attgatgatt cctttccgct actaatatca cagtcctcac ggcaagaacc caattccatg ggctggatcc tcaaaaagaa aatcagtggg cgatagaaac ggtctatcag aagaagtttc gtgtcattca ttacacgccc tgtgtaattc taacatgaat ttggtggaaa gattctagca atatatatta tttgaagcac aactatatga cagaattata gacgacgatg gatgtggaag gtcctcacta gcttcttttg

9919 ttgcgtgtag ggactggtgt agaaggacga cgtcgggcag tagttggtcc taatttgcag aacagcagag ggcagcagaa aactaattgg tatgagcttt ggcgtttcca tagtggattc tcctcaggac taccaaagaa gagagaaggt ccggacatag ttgggtctat atatttttgg acaagtcaga ggactcagaa ctctggctca agaagatttg tgacagcgaa catgtatgca caaatgttgg actcagtgtt gagttacaaa gttgccagaa atatccaaca actctgtaac caaatctgta acctgcagca cgccaacgat tggtacagca agagagttta aagtcgaatc aaggccttct tagtcgtttc ggttgtgttt tccattagag gtatacgttt attgaataga acatacttgt cagaagagat ttgaaaagtt caggaagtaa ctgcatttca tccctaatcc cagtccattg aaaagacttg tcgacgctga gtgtccactg aacacaaagg ttgctgttat cacaggttct agactacaac aggaagaatt gctcggaaat tgaccagtgg agggcaattt cgcacctgca cccagaaatg atgcaatgga ctcttatctc aggttaaagg gttcagggaa gatgaacaat accatcatgg taccattggt ataatacaca agacataaac aaaattacac cggttagacc tgtcagggac ttagtgtctc tcagcagtgt taaagatagg ggcagcaaaa aaatgcatca agaacatgga tgttctatta tgagtgtctg tggtgacatg tcaaagatca ctcatctaat tgaatttgca aagggtttca aacggcaagt tgattgcttt atagttggcg aaaga cgttt taacgaaccc gtctggattg ctggtgagcc tgattcctac ggttagccca tcggagatat gtgaaagctt catgtgggaa gtgcaaagga gctgactagg tagacctgaa aagaattagg cttgaatatt ttgacattat gttacattgttcg tcaaa

FIG. 14a gatgagctga gtgtgtagta gatggaaatg cttgttaaga agccggaaaa atcagggtac cttaaaactt gagtttcaga gggctacttg tccttcggtg acactgtaga acoctatgaa ttcccgcttg ttggtttctc gcctcgtctc ggatcactgt tttttgctta ttgtgaagca ataaaggaca attccctcaa ggagtcatga tcagtctctt gtttcaatcg ttgttaactt agaatgcacc actaacttgc aagatacata cttttggttc ttcacacttt atgtacataa acaacatttg cgaagagcga atcatgaaaa gagtctcacc ggttgatttc attccaaatt atgtaccttt gcaagccagt

187 851

Fig. 14b tgagtcaagt cctcacagtt cataatctgg tcgagcactg ccgaacagcg cgggaagaat cgtttcccga gttccactga tgataaaaaa aacaaggtca gacagcaagt aacaaaacca tgtttaaaga tcatttagtt ttgttttttg tgataaggag tccgatgaag tgggtgagaa tccataccgg ttttagaaag cgcttttagt ctactttgat gctcttctag gattctgaaa ggtgctatct ttacacccgg tgatgttctc ttcgtaccag tgagacggtc aggctcgagg ctagtcacta tgaactcaca tgttcccttc atttcggcga tctccattgc agcttgtgct tccgttggaa aaagacgttg agcaagtgca actaaacagt ggacgacaca aagaatagtt atcattagtt cactcagttt cctaatagag aggacataaa tttaattcaa acatataaga aataagactt gatagatacc tctattttca agatcgagca gcgtcatctt caattcatcg gccgccactg caaaagaggg aggaacatct ctaggaattt gttctcgttt gtcttcttgc tctagtattt ctacacatag tcggcctttg agagaatgct tgcattgctc cgggatatta ttacattcaa ccgccatagt ggcttgtttt gcgatcatga gtgcggttct accttccaaa gttgcttctg atgcacttgc acctttttcc aatagagata gtatcaattg tggctccttc cgcatcgcag caacatgaag caccgtatat cccctcggat tcctatggtt gacatcggca agatcaagtt ttaaaagatc tgttgcggtc ttcacattgc aatatgcaac agcgaaatga and gagcacacg catcatctag attggtgtga tcctctttca aaagcaactt gactaactca atatcatccg agtcaagtgc cttatgtaca ttcgagacat gtttctttac tttaggtacc tccaaaccaa gctctttacg tctatcaatt atctctttaa caagctcttc cggcaatgac ttttcaagac taaccatatc tacattagac ttgacaataa tctctttaca tctatccaat agcttcatac aagctttacc acatatatta gcaagcttga gtataaccaa tgtgtcctct ataacaactt tgtctacaac gtccaataag tgcctctgaa atacaaatac aagtactcaa gtaagaacat attcatgaat gtgtaaccat agcttaatgc agatggtgtt ttacctgata gagagtaatt aattcaggga tcttgaagat gaaagccaaa tagagaacct ccaacatgaa atccaccgcc ggccggcaag ccacgtggca gcaattctcg tctgcgcatt cagaaactcc tttaggcggc ggtctcactc tgctgctgta aacataagcc aaaacagtca caaccgaatc gaaaccgact tcgtaatcct tggcaatctc cttaagctcg agcttcacgg cggcggtgtt gttggagtct ttctccttct tagcggcggc taaagcgctc ttgaagaaag agcttctcgc tgacaaaacg caccggtgga aagaaacttc ccggccgtcg gagagaacaa gcttagcgtc gctgtagaaa tcatccggcg agtcaaagac ggattcgaag ctgttggaga gcaattgcag agcagataca tęaggtcęgg tgagtacttg ttcggcggcc agataaacaa tagaggagtc ggtgttatcg gtagcgacga aactagtgct gctgatttca taagaatcgg cgaatccatc aatggtggtg tccatcaaca ggttccgatg aattgaaatt cacaaattaa agagatctct gctaatcaac gaagagacct tatcaactgg

187 851

FIG. 14c atttggttaa agatcgaaga taaccattga cgagcagagc caagtcaagt caacgagagt ggtggtgaga tatgaagaag catcctcgtc ccacggttta catttcacca aaaccggtaa atttccagga aaggaatctt tgtcagagat cttttttaaa aagatataac aggaagctaa accggttcgg gttataaatg ttagtattta taccggagac attttgtgtt gctaattttt gtatatgaga agttcaatcc ggttcggtaa gcccctgaac caaactagat ttggagatga tataaatata taaaatttat ttttcatccg gttcgttatt ttcatataaa tatacaaata ttatttttta aatttaagaa ttagatttac atgtgaaagt tacatttctg tttattttct ttgaagtaaa atgataaagg gaacgtatat taagtttcat gctttattca cataagtttt gtaatgtata ttatattttt cgtttattga aaaagtaatt ttcagtgttc agcatgttta cactataatt aaatcaagtc gaatatttcc tggaactatt ctccttgttc tatagcaaat gaaaacgctc ttcacaacaa aatcattata gatataggaa taaattacat taaaaacatg aaagtcataa tgaatatatt tttttaatta ggatttgaCt taaaaacaat tattgtatac atataaaaga cttctttagt tatttgcctt caacttctcg ttctgaatca tgcgataaat cagctttttc aataactacg acgtaaaagc aaattcataa cacgtctaaa caaatttggc tcatccttca cttgattggt gttttccgga ctcgatgttg ctggaaactg agaagaagaa g gaatctgca taatcacctc ttggttcctc accggtagac tcattttgtt ggatcgaaaa cgatcgagat cagaaaatga aaagataggt taaagatgcc tatgaataca acaacgtaag attatgttga ataaacagag tactttatat aggagttata ataaggtaaa taaattattg ctttccgcgt tttttacttt tgtatttctt aaatgataag ttaaattagg ataagatttg tatgatttta agtaaattta caataactct ctataactca atagcatcac atatttaatt aattttacta attatctttt gaacaatttt atgaaatagt tttcttttaa ttaatttttt aaaatgatat attataaaat ttaattgaat caatctgata taattttttt atcttctacc atctattata gttgataaat attgtgataa actttagata aacacccaat tgccaaatat ttaataaatt ttgtgtacca tgcgtttttt ttggagaata tatatacgtg gacagcatac cgtacatata ttgtataaaa gcttataaaa catagatacg ggttatattg gtaagctata aatatatgta aacaatagta agatattacg tgttgtgtct aaatatgtgt tgctttagat attatgtata tctaatatat taaaatatct tttattaact aatatattat ttaagagaga aaattgggac actattttct atacagtaac tgttttcaac tataaacagg aacccttgat ataataaaat aactagccaa aaaatcagat taaatattca taaaacaątg tttggtatta ttacataaac ctaagaaaca aaattcaata ttccttttta ccttataaaa aacaattaaa catcactaga tatatttatg ccccacaatg agcgagccaa ttgagacttg agacttgaga tccttgtcaa ctacgtttgc atttgtcggc ccattttttt tatttttttt ttaaagtgtc ggcccgttgc ttcttccgtt cagatcaacc

187 851

FIG. 14d ctctcgtaat cagaacaaaa cggaaaacaa acgaaagaac aatcagatcc ctcttttttt gcataaacta aattcaactt ctctgcgttt atgttgtaga ggcaaccacg atcactacta cgaaacaata caacgtcgtt gcttggagtc cacgtaatca aatctactcc aatgctttta atatctttca ctttaaccca cgacttttca aaactgctct ttaaaaccca taactcgtga acatcttctt gatctttgtt tgtccactga cgaatagcac ctagcttccc ttcgtatctg actaatcctg agaaaacatc agagttcgga gtatggaaga aggaccaagt ttcggttttg agacaaaacc ggatcacatt gttgttccgt gatatccaat gcaagaaccc cgaaacttgt atcgggttgg aaaaaattaa tctgtctgtt tttggtagac gcaaattttc taatctcttc caggtaaacg aatcagaatc gaaaacttcg cacataaaag ttctgtgatt caaatggtag ataccccgag acatacacat acgccgagac tgcgaaagcc tttgtatttt ataccggaaa gggttcaatc cgattaccgc taaacccaat gacatatccc aacccttcac ttctggcttt ggtatgacct gatactgttt agtggttggt ttgaagacta tgtatccacg tgatggtttt gtatacttaa cacaaagcaa tatcccatga cttgcatcac aagcttcgat ctttatcatt ccgggtggca gaaagtcgat ggagactcca ttgttttgta aatcactcct ctcatggaca aaactggttc gaagttcgtg tccttttact atgtagtgtt gtatgaagta tcccgaaata c gattggttc taaggagatt aagattgaca aaccatgact cgtagcttct cttgttgcac tctttattca ggagcctgaa ttttccgatt tttgacgccg gaagataaga aagaaattct tggatcatgt cttgatttat caccggagaa ctcatgatcc tgtcgggaat aaagagatga gcacgatcac tgaatgagaa atgaaaaaat caggatcggt agagaacaac ttatgatgaa taaagtgttt atatatcctt tctttgttta aggaaagtat caaaatttgc ctttttcttc gctagtccta aaacaaacaa attaaccaaa agataaaatc tCtcatgatt aatgttactt gtgataccte aagccaaaac tttatcttta gacttttaac caaatctaca gtaatttaat tgctagactt aggaaacaac tttttttttt acccaacaat ctttggattt taattgtttt tttttctact aatagattaa caactcatta tataataatg tttctatcat aattgacaat tctttctttt taataaacat ccagcttgta taataatcca caagtcaatt tcaccatttt ggccaattta ttttcttata aaaattagca caaaaaagat tatcattgtt tagcagattt aatttctaat taacttacgt aatttccatt ttccatagat ttatctttct ttttatttcc ttagttatct tagtactttc ttagtttcct tagtaatttt aaattttaag ataatatatt gaaattaaaa gaagaaaaaa aactctagtt atacttttgt taaatgtttc atcacactaa ctaataattt tttttagtta aattacaata tataaacact gaagaaagtt tttggcccac acttttttgg gatcaattag tactatagtt aggggaagat tctgatttaa aggataccaa aaatgactag ttaggacatg aatgaaaact tataatctca ataacataca tacgtgttac tgaacaatag taacatctta cgtgttttgt

187 851 ccatatattt gcttttaaga accaaaaaaa cctacgttga tccgtccaga aagaggcaaa aagttgtaga gcttggtatc ctttttccca acatcttctt ttcgtagctg aagaccaagt gatctccaat accgtctgtt aatcggaatc ccaggcgatt ggttaccgtt gtatgatctg attggtcttg agctttgatc tgttatccaa cctctttcgt gagatttagg tattgatgag acctcttgga ggaaaaaaga aatcaggatt gtatcacaaa caaagataaa taaaaatata caaagtttct gacttagaaa caatctttgg ctcattatat tttaaaaatt attaaactta ttagtacttt actccagttt gtaataaata gttgcttata agcacaaaac aaaattaaat gccttgttga tcaaccctct atccttgttt ggcaaacatg cacgtaatat cgacttttca gattgttgtt actaactctg ttcggttttg gcaagaaccc ctcggtagac aaaaacttcg tcgaaagcct agacctaatg atactgtttt tatcattata tttacctctc atctctcctc agcgttctag ttgacaaacc cctcaatttt tcgtgtcctg aagaaagaga ggtagagaac ttgccttttt atcttcatta tataaaatat cttttgactt acaacatttt gtttctatag catatacaaa agcaagtttg gtgatttcca aaattttcat aacttatgtt ttttagcttt aatatattca catataacaa attcctacag ctagtttgca cgtaatcaga gtatgaacta atcccaacta gctctactcc aaactgctct tatccagtga ggaataaacc ggacataacc tg aagcttgt gcaaattttt cacgcaaaag aaatatttta acttatcaca gttgttggtt acaaagcaat cttgtggcag tcatggaaaa gaagtatcca aagactcgta ccgatttcgg atttatcacc tgagcacgat cgacgatgat cttcgctagt atgttttcct ttcattaggt ttaaccaaat gtatatatat tgtttttttt gtgtttcctt ttcttatatc ttttgcacct atatataatt aaatgtttca atgaaaaaaa tataacaatg aattaaatat ccttgttgat tttgtcggtc acaaaagggg agtttaactt gaaagcatta aatgctttca ttaaaaccca cgaataacac aacgtttgga ggatcacatt accgggtttg taatctcttc ttctgagatt taccggaaag actcctcact tgcagactat atcccatgac aaaatcgatg aactggtatc gagatattgt gcttctcttg acccccgaag ggagaactca cagtgaatga gaatatacaa cccaaaacaa ttttcttcgc tacttgtagt taacagtaat tctttgacat cttattctaa ttcaatcaac atcattcagc atatgtttct tattaaaatt tcacactaaa atatcaaatc

FIG. 14u tttgcattaa tcctatccct tattttatgc catttcttct aaacaaacgt ctctgtgttt cgacgtcgtt atatctttca taatctgtga ctagcttccc gtatgtaaga gtggttccat aaagaattag cacataaacg ccgagtcata gctgcaatcc tttgggtttg gtattccggt gtgcatcaca gagactcctt aagtttgtat tggttcgatg ttgcactctt ataagaaaga tgatcttatt gatatataga gtgtttataa gcaaattaac cagtcccaga accttgagcc ttaatagcta caaaattcaa tagattacca atccattttc agatttctta ctttcttagt aaaagtaaaa agagcattaa actgaagaca

187 851

9701

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Fig. 14f aatagactaa atattctgat atttcacaca gattcttgta

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187 851

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187 851

C SA INA BTH EmWa

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them'3

them-4

them-5

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187 851 c SA INA BTH EmWa

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187 851 o λ <o s Q QQO Q ca b □ i

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187 851 οιοωΐ'-νΩ Ω Ω Ω Ω ϋ AB ίΐ □ Β ig. 18A3

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187 851

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187 851

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Fig. 18B2

187 851

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187 851

Fig. 19

him: 267 VSNVHKALDSDDIELVKLLLKEDHTNLDDACALHFAVAYCN 307 Rice-1: + + + ALD + DIELVKL ++ + + LDDA A + H + AV + CN 33 IRRMRRALDAADIELVKLMVMGEGLDLDDALAVHYAVQHCN 155 him: 327 PRGYTVLHVAAMRKEPQLILSLLEKGASASEATLEGRT 364 P G T LH + AA P ++ LL + A + T + G T Rice-1: 215 PTGKTALHLAAEMVSPDMVSVLLDHHADXNFRTXDGVT 328 him: 267 VSNVHKALDSDDIELVKLLLKEDH! WLDDACALHFAVAYCN 307 Rice-2: + + + ALD + DIELVKL ++ + + LDDA A + H + AV + CN 33 IRRMRRALDAADIELVKLMVMGEGLDLDDALAVHYAVQHCN 155 him; 325 RifPRGYTVLHVAAHRKEPQLlLSLLEK 351 R P T LH + AA P ++ LL ++ Rice-2; 208 RRPDSKTALBLAAEMVSPDMVSVLLDQ 288 him: 267 VSNVHKALDSDDIELVKLLLKEDHTNLDDACALHFAVAYCN 307 + + + ALD + * DIELVKL ++ + + LDDA A + H + AV + CN Rice-3: 33 IRRMRRALDAADIELVKLMVMGEGLDLDDALAVHYAVQHCN 155 him: 325 RNPRGYTVLHVAAMRKEPQLILSLLEK 351 R P T LH + AA P ++ LL ++ Rice-3: 208 RRPDSKTALHIAAEMVSPDMVSVLLDQ 288 him: 267 VSNVHKALDSDDIELVKLLLKEDHTNLDDACALHFAVAYCN 307 Rice-4: + + + ALD + DIELVKL ++ + + LDDA A + H + AV + CN 33 IRRMRRALDAADIELVXLMVMGEGLDLDDALAVHYAVQHCN 155 him: 327 PRGYTVLHVAAMRKEPOLI 345 P G T LH + AA P ++ Rice-4: 215 PTGKTALHLAAEMVSPDMV 271

187 851

Molecular phenotype of a mutant them

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SA INA

I-1 Iμ ε +, ε c 5 c

BTH

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PR-1

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Fig. 1

PR-5

Publishing Department Uh Rh. Circulation of 70 copies. Price PLN 6.00.

Claims (18)

Patent claims 1. An isolated DNA molecule capable of conferring disease resistance in plants, encoding the amino acid sequence of the NIM 1 gene shown in SEQ ID NO: 2. 2. An isolated DNA molecule according to claim 1 according to claim 1, which comprises the nucleotide sequence shown in SEQ ID NO: 2. An isolated DNA molecule according to claim 1 or 2, characterized in that it comprises a nucleotide sequence further shown in SEQ ID NO: 1. 4. Plasmid BAC-04, ATCC deposit No. 97543. 5. A chimeric gene containing a plant active promoter operably linked to a heterologous DNA molecule, characterized in that the heterologous DNA molecule is a molecule identified by a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2. 6. The chimeric gene of claim 1 The method of claim 5, wherein the heterologous DNA molecule is defined by the nucleotide sequence shown in SEQ ID NO: 2. 7. A recombinant vector which comprises a chimeric gene according to claim 5 or 6. 8. A recombinant vector according to claim 1 The method of claim 7, capable of stably transforming a plant, plant seed, plant tissue, or plant cell. 9. A plant expression cassette containing a chimeric gene as defined in claim 1. 5 or 6. 10. A plant expression cassette according to claim 1. 9. The chimeric gene is expressed in a continuous or constitutive manner therein. 11. A plant cell, characterized in that it comprises the chimeric gene according to claim 1. 5 or 6. 12. A plant cell having a broad range of disease resistance, characterized in that it comprises the chimeric gene according to claim 1, 5 or 6. 13. Plant cell according to claim 1 The composition of claim 11, wherein it is derived from a plant belonging to the group consisting of gymnosperms, monocots and dicots. 14. The plant cell according to claim 1 11, characterized in that it comes from a useful plant. 15. A plant cell according to claim 1 14. characterized in that it is derived from a plant belonging to the group consisting of rice, wheat, barley, rye, corn, potato, carrot, cavity, sugar beet, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnips, radishes , spinach, asparagus, onion, garlic, eggplant, peppers, celery, carrots, squash, pumpkin, zucchini, cucumber, apples, pears, quinces, melon, plums, cherries, peaches, nectarines, apricots, strawberries, grapes, raspberries, blackberries , pineapple, avocado, papaya, mango, banana, soybeans, tobacco, tomato, sorghum and sugarcane. 16. Use of an isolated DNA molecule as defined in claim 1 1-3, in screening assays to identify compounds capable of inducing a wide range of disease resistance in plants. 17. Use of an isolated DNA molecule as defined in claim 1-3 for imparting disease resistance to plant cells, plants, and their progeny. 18. Use of resistant plant cells as defined in claim 1, 12 to incorporate a resistance trait into a plant line by crossing. * * * 187 851