BRPI0813360B1 - ISOLATED POLYNUCLEOTIDE, VECTOR, PROCESS FOR TRANSFORMING A HOST CELL, PROCESS FOR PRODUCING A PLANT, PROCESS FOR CONFERRING OR IMPROVING RESISTANCE TO COLLETOTRICHUM, PROCESS FOR ALTERING THE LEVEL OF EXPRESSION OF PROTEINS CAPABLE OF CONFERRING RESISTANCE TO COLLETOTRICHUM AND BEAM ROT LE OF A PLANT CELL AND IDENTIFICATION PROCESS OF A CORN PLANT - Google Patents

Abstract

ISOLATED POLYNUCLEOTIDE, VECTOR, RECOMBINANT DNA CONSTRUCTION, PROCESS FOR TRANSFORMING A HOST CELL, PLANT CELL, PROCESS FOR PRODUCING A PLANT, PLANT, SEEDS, PROCESS FOR CONFERRING OR IMPROVING RESISTANCE TO COLLETOTRICHUM, PROCESS FOR DETERMINING THE PRESENCE OR ABSENCE OF THE POLYNUCLEOTIDE, PROCESS TO ALTER THE EXPRESSION LEVEL OF PROTEINS CAPABLE OF PROVIDING RESISTANCE TO COLLETOTRICHUM AND ROT IN THE STEM OF A PLANT CELL, PROCESS OF IDENTIFICATION OF A CORN PLANT, SEED DESCENDANT OF A DESIGNED VARIETY OF CORN. This invention relates to gene-encoding polynucleotide sequences that can confer resistance to the plant pathogen Colletotrichum, which causes anthracnose stalk rot, leaf spot and death of corn and other cereals. Furthermore, it refers to plants and plant seeds with chimeric genes comprising said polynucleotide sequences, which enhance or confer resistance to the plant pathogen Colletotrichum, and processes for producing said plants and seeds. The invention also presents sequences that can be used as molecular markers, which in turn can be used to identify the region of interest in corn lines resulting from new crossings and quickly and efficiently introduce the genes (...) .

Description

Field of Invention

This invention relates to compositions and methods useful in creating or improving resistance to pathogens in plants. Additionally, the invention relates to plants that have been genetically modified with the compositions of the invention.

Background of the Invention

Colletotrichum graminicola (Ces.) (CG), better known as anthracnose, is the causal agent of anthracnose leaf spot, anthracnose stem rot (ASR), and apex decay affecting Zea mays (L.), also known as corn or cereal. This is just the commonly known stem rot that also causes leaf spot (Bergstrom, et al., (1999), Plant Disease, 83:596-608, White, D.G. (1998), Compendium of Corn Diseases, pp. 1 -78). It has been known to occur in the United States since 1855 and has been reported in the Americas, Europe, Africa, Asia, and Australia (McGee, D.C. (1988), Maize Diseases: A Reference Source, for Seed Technologists, APS Press, St. Paul, MN , White, (1998) supra; White, et al., (1979) Proc. Corn Sorghum Res. Conf (34th) 1-15). In the United States, more than 37.5 million hectares are infested each year, with yield losses averaging 6.6% nationally (see Figure 1). Yield losses are due to both low grain weight in infected and "bedded" plants, that is, the drooping of plants due to weakness in the stalks caused by the infection (Dodd, J., (1980), Plant Disease, 64: 533-537). Lodging plants are more difficult to harvest and are susceptible to other diseases. After infection, usually the upper part of the stem dies first while the lower stem still remains green. Externally, the infection can be recognized by the mottling of black spots on the outer bark of the stem, while internally the pith tissue is discolored or black in appearance. Inoculation occurs in several ways. Roots can grow through stem fragments and become infected. This will be a growing problem as "no-till" methods of agriculture are more widely adopted due to their environmental benefits. The fungus can also infect stems through insect damage and other injuries (White (1998) supra). Stem infection may be preceded by leaf infection causing leaf spot and providing inoculum for stem infection. There is controversy in the technical literature regarding the number of different varieties or races of Cg present in nature. The pathogen is transmitted by wind or contaminated seed lots. Spores remain viable for up to 2 years (McGee (1988) supra; Nicholson, et al., (1980), Phytopathology, 70:255-261; Warren, H.L. (1977), Phytopathology, 67:160 - 162; Warren, et al., (1975), Phytopathology, 65:620-623).

Farmers can combat infection with corn fungal diseases such as anthracnose through the use of fungicides, but these have environmental side effects and require field monitoring and diagnostic techniques to determine which fungus is causing the infection so that the correct fungicide can be used. to be used. Particularly with large fields of crops like corn, this is difficult. The use of corn lines that carry genetic or transgenic sources of resistance is more practical if the genes responsible for resistance can be incorporated into elite, high-yield germplasm without reducing production. Genetic sources of resistance to Cg have been described. There have been several corn lines identified that carry some level of resistance to Cg (White, et al. (1979) supra). These included A556, MP305, H21, SP288, CI88A and FR16. Reciprocal translocation crossover test analysis using A556 indicated that genes controlling resistance to ASR rest on the long arms of chromosomes 1, 4, and 8, as well as on both arms of chromosome 6 (Carson, ML (1981), Sources of inheritance of resistance to anthracnose stalk rot of corn. Doctoral Thesis, University of Illinois, Urbana-Champaign). Introgression of resistance from such lineages is complex. Another natural, LB31, has been reported to carry a single dominant gene controlling resistance to ASR, but appears to be unstable, especially in the presence of European corn borer infestation (Badu-Apraku et al., (1987) Phytopathology 77: 957- 959). Strain MP305 was made to carry two dominant genes for resistance, one with a greater effect and one with a lesser effect (Carson (1981) supra). MP305 was made available by the University of Mississippi through the National Plant Germplasm System (GRIN ID: NSL 250298) operated by the US Department of Agriculture. See Compilation of North American Maize Breeding Germplasm, J. T. Gerdes et al., Crop Science Society of America, 1993. MP305 seeds can be obtained from W. Paul Williams, USDA-ARS Genetic Research Supervisor, Corn Host Plant Resistance Research Unit, Box 9555, 340 Dorman Hall, Mississippi State, MS 39762.

It has been reported that there are two genetically separable genes (meaning they behaved as separate genetic loci) linked on the long arm of chromosome 4 that confer resistance to Cg (Toman, et al., (1993), Phytopathology, 83:981 - 986 ; Cowen, N et al. (1991) Maize Genetics Conference Abstracts 33). A significant resistance quantitative trait locus (QTL) on chromosome 4 has also been reported (Jung, et al., (1994), Theoretical and Applied Genetics, 89:413-418). Jung et al. (supra) reported that UMC15 could be used to select QTL on chromosome 4 in MP305, and suggested that the QTL is in a 12cm region of chromosome 4 between UMC15 and UMC66. Indeed, as discussed in greater detail below, the region between UMC15 and UMC66 as reported in genetic map 4 of IBM2 neighbors is approximately 129 cm, and selection for the QTL in the manner suggested by Jung et al. (1994, supra) would be the best way to select a large chromosomal interval with considerable linkage hindrance and negative phenotypic effect, and in the worst case, a recombination could occur between the two markers, resulting in a selection of false positives for the locus Rcgl. The region carrying the genes responsible for the phenotype conferred by the QTL on chromosome 4 will be referred to here as the Rcgl locus or the MP305 resistance locus; this has been referred to as the ASR locus.

Much work has been done on disease resistance mechanisms in plants in general. Some resistance mechanisms are non-pathogen specific in nature, or so-called "host non-resistant. These may be based on cell wall structure or similar protective mechanisms. However, while plants lose an immune system with circulating antibodies and others attributes of a mammalian immune system, they have other mechanisms to specifically protect against pathogens. The most important and best studied of these are plant disease resistance genes, or "R" genes. The R genes can be found in Bekhadir et al., (2004), Current Opinion in Plant Biology 7:391-399. There are 5 recognized classes of intracellular proteins with one nucleotide binding site (NBS) and one. leucine-rich repeat (LRR); transmembrane proteins with an extracellular LRR domain (TM-LRR); transmembrane LRR with a cytoplasmic kinase domain (TM-CK-LRR); membrane signal-anchored protein with a coiled-coil cytoplasmic domain (MSAP-CC) and membrane-associated kinase with an N-terminal myristylation site (MAK-N) (see, for example: Cohn, et al., (2001), Immunology, 13:55-62; Dangl, et al. (2001), Nature, 411:826-833).

Broglie et al. (US Patent Application Serial No. 11/3 97, 153) described a new R gene related to the NBS-LRR type designated Rcgl found within the Rcgl locus previously described by Jung et al. (above). They described useful markers in crosses with this locus, chromosomal intervals that can generate maize plants with resistance, and transgenic plants containing the Rcgl gene. The present invention improves on the work of Broglie et al. providing a second NBS-LRR gene, different from the Rcgl gene and designated Rcglb, which is required in combination with Rcgl for Cg resistance. The Rcglb gene is physically (KB 200-300) and genetically (less than 1 centimorgan) closely linked to Rcgl and represents a second component of the Rcgl locus. The present invention provides Rcglb sequences, chimeric constructs combining Rcgl and Rcglb, and methods and markers for production with the Rcglb and Rcglb genes together (the Rcgl locus).

Summary of the Invention

Embodiments of this invention are based on the fine mapping, cloning and characterization of genes responsible for the resistance phenotype of the MP305 strain, the introgression of a truncated chromosomal interval with the MP305 resistance locus (Rcgl locus) in strains with little or no linkage drag, the demonstration of the use of these genes as transgenes and the use of molecular markers to move genes into elite lines through breeding techniques.

Embodiments include an isolated polynucleotide comprising a nucleotide sequence encoding the Rcglb polypeptide, which together with the Rcgl polypeptide, confers resistance to Colle totrichum, wherein the Rcgl polypeptide has an amino acid sequence of at least 50%, at least 75 %, at least 80%, at least 85%, at least 90%, and at least 95% identity when compared to SEQ ID NO: 3 or the peptide encoded by the Rcgl sequence deposited with the Agricultural Research Service Culture Collection (ARS) on February 22, 2006 as Patent Filing in NRRL B-30895, and further that the Rcglb polypeptide has an amino acid sequence of at least 50%, at least 75%, at least 80%, at least 85 %, at least 90%, and at least 95% identity when compared to SEQ ID NO: 246 or the peptide encoded by the Rcglb sequence deposited with the Agricultural Research Service (ARS) Culture Collection on June 26, 2007 as Patent Application No. 2 NRRL B-50050 based on the Needleman-Wunsch alignment algorithm, or a complement of the nucleotide sequence, in which the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary. Embodiments also include an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide capable, together with the Rcgl polypeptide, of conferring resistance to Colletotrichum, wherein the polypeptide has an amino acid sequence of at least 50%, at least 75 %, at least 80%, at least 85%, at least 90%, and at least 95% identity when compared to SEQ ID NO: 246.

Additional embodiments of the present invention include a vector comprising the polynucleotide encoding a polypeptide of an embodiment of the present invention, such as SEQ ID NO: 3, SEQ ID NO: 246, or the polynucleotide sequence of the plasmid deposited as Patent Deposit No. 2 NRRL B -30895, or the polynucleotide sequence of the plasmid deposited as Patent Deposit in NRRL-B-50050, and a recombinant DNA construct comprising the polynucleotides of an embodiment of the present invention operatively linked to at least one regulatory sequence. Additional embodiments of the present invention include vectors comprising polynucleotides encoding both SEQ ID NO: 3 and SEQ ID NO: 246. A plant cell, as well as a plant, each composed of a recombinant DNA construct of an embodiment of the present invention, and a seed comprising a recombinant DNA construct of the embodiments is also incorporated by the present invention.

The methods embodied in the present invention include: 1) a method for transforming a host cell, including a plant cell, comprising transforming the host cell with the polynucleotide of an embodiment of the invention, 2) a method for producing a plant comprising transforming a plant cell with the recombinant DNA construct of an embodiment of the present invention and regenerating a plant from the transformed plant cells, and 3) methods for conferring or increasing resistance to Colletotrichum and/or stem rot, comprising transforming a plant with the construct of Recombinant DNA of an embodiment of the present invention thereby conferring and/or improving resistance to Colletotrichum or stem rot.

Additional embodiments include methods for determining the presence or absence of polynucleotides of an embodiment of the present invention, or the Rcgl locus, in a corn plant, comprising at least one of (a) isolating nucleic acid molecules from the corn plant and determining whether an Rcgl gene is present or absent, and determining whether an Rcglb gene is present or absent, by amplifying sequences similar to the polynucleotide, (b) isolating nucleic acid molecules from the corn plant and performing Southern hybridization, (c) isolating proteins from the corn plant and performing a Western blot using antibodies against the Rcgl protein and/or the Rcglb protein, (d) isolating proteins from the corn plant and performing an ELISA test using antibodies to the Rcgl protein and/or to the Rcglb protein, (e), demonstrating the presence of mRNA sequences derived from the Rcgl mRNA transcript and/or the Rcglb transcript exclusive to Rcgl and/or Rcglb, or (f), demonstrating the presence of newly conferred or improved resistance to Colletotrichum infection through a phenotypic assay, thus determining the presence of polynucleotides or the Rcgl locus in the corn plant.

Methods for altering the expression level of a protein capable of conferring resistance to Colletotrichum or stem rot in a plant or plant cell comprising (a), transforming a plant cell with the recombinant DNA construct of an embodiment of the present invention and ( b) cultivating the transformed plant cell under conditions that are suitable for expression of the recombinant DNA construct where expression of the recombinant DNA construct results in the production of altered levels of a protein capable of conferring resistance to Colletotrichum or stalk rot in the transformed host are also incorporated by the present invention.

An additional method incorporated by the present invention is a method of assigning or improving resistance to Colletotrichum and/or stalk rot in corn plants, comprising (a) crossing a first corn plant lacking the Rcgl locus with a second corn plant containing the Rcgl locus to produce a segregating population, (b) analyzing the segregating population for a member containing the Rcgl locus with a first nucleic acid, not including UMC15a or UMC66, capable of hybridization with a second nucleic acid linked to or located within the Rcgl locus and (c) selecting the member for further crossing and selection.

Methods for increasing resistance to Colletotrichum and/or stalk rot, or introgressing Colletotrichum and/or resistance to stalk rot into a corn plant, comprising representing a corn plant assisted selection marker with a nucleic acid marker, in that the nucleic acid marker specifically hybridizes to a nucleic acid molecule with a first nucleic acid sequence that is linked to a second nucleic acid sequence that is located at the Rcgl Locus of MP305 and selecting the corn plant based on the marker. assisted selection are also embodiments of the present invention. Specific FLP, MZA and specific Rcgl SNP markers disclosed herein are other aspects of the invention.

Another embodiment of the invention is a process of identifying a corn plant that exhibits improved or newly conferred resistance to Colletotrichum infection by detecting alleles of at least one marker in or within the Rcglb gene and, optionally, by detecting a second marker on or within the Rcgl gene.

Further embodiments include methods of identifying corn plants that exhibit improved or newly conferred resistance to Colletotrichum by detecting alleles of at least 2 markers in the corn plant, in which at least one of the markers is at or within the chromosomal interval below UMC2 041 and above the Rcglb gene, and at least one of the markers is at or within the range below the Rcgl gene and above UMC2200. Similar embodiments covered by this process include at least one of the markers being at or within the chromosomal interval below UMC1086 and above the Rcglb gene, or within the chromosomal interval below UMC2285 and above the Rcglb gene, and at least one of the markers is in the or within the range below the Rcgl gene and above UMC2200, at or within the range below the Rcgl gene and above UMC2187, or on or within the range below the Rcgl gene and above UMC15a.

Embodiments further include processes of identifying corn plants that have recently attributed or improved resistance to Colletotrichum by detecting alleles of at least 2 markers in the corn plant, in which at least one of the markers is on or within the chromosomal interval below UMC2041 and above the Rcglb gene is selected from the markers listed in Table 16, and at least one of the markers at or within the range below the Rcgl gene and above UMC2200 is also selected from the markers listed in Table 16. Embodiments include processes of identifying corn plants that have recently attributed or improved resistance to Colletotrichum by selecting at least four markers or at least six, wherein at least two or three of the markers are at or within the chromosomal interval below UMC2041 and above the Rcglb gene, and at least two or three of the markers are at or within the range below the Rcgl gene and above UMC2200. Additional embodiments include this same process when the two or three markers at or within the chromosomal interval below UMC2041 and above the Rcglb gene, as well as the two or three markers at or within the interval below the Rcgl gene and above UMC2200, are selected. from those listed in Table 16. Another embodiment of the process includes detection of allele 7 in MZA1112, detection of allele 2 in MZA2591, or detection of allele 8 in MZA3 43 4. Corn plants and seeds produced by established processes are also embodiments of the invention, including corn plants that do not contain the same alleles as MP305 or equal to or above UMC2041, or equal to or below UMC2200 at the loci shown in Table 16.

Other embodiments include processes of identifying corn plants that have recently attributed or improved resistance to Colletotrichum by detecting alleles of at least 2 markers in the corn plant, wherein at least one of the markers is on or within the chromosomal interval below UMC2041 and above the Rcglb gene, and at least one of the markers is at or within the range below the Rcgl gene and above UMC2200, and where the process detects the presence or absence of at least one marker located within the Rcgl gene or of the Rcglb gene. This new embodiment includes a modification of the process in which four markers are selected for, wherein two of the markers are within the chromosomal interval below UMC2285 and above the Rcglb gene, and at least two of the markers are within the interval below the Rcgl gene. and above UMC15a. Further embodiment of this process includes the Rcgl gene and the Rcglb gene having been generated from a donor corn plant, including MP305 or DE811ASR (BC5), into a recipient corn plant to produce a generated corn plant. This process also includes the instant when the generated corn plant line is selected for a recombination event below the Rcgl gene and above UMC15a, so that the generated corn plant line maintains a first MP305 derived from the chromosomal interval below the gene Rcgl and above UMC15a, and do not maintain a second MP305 derived from the chromosomal interval below UMC15a. Corn plants and seeds produced by these processes are also embodiments of the invention. Corn-generated lines incorporated by the invention include those that are conversions of the Rcgl locus of PH705, PH5W4, PH51K or PH87P, or their descendants.

A further embodiment of the invention is a process of identifying a corn plant that exhibits greater resistance to Colletotrichum infection, by detecting in the corn plant the presence or absence of at least one marker at the Rcgl locus, and selecting the corn plant in that at least one marker is present. Embodiments include when at least one marker is at or within SEQ ID NOs: 137, 255, 256, 257, 258, or 266 and also when at least one marker is at or within the coding sequence of Rcgl or Rcglb, or located on or within the polynucleotides contained in SEQ ID NO: 1 and SEQ ID NO: 245.

Generated lines of corn plants embodied by the invention include those that are conversions of the Rcgl locus of PH705, PH5W4, PH51K, or PH87P, or descendants thereof. Such embodiments include corn seeds that comprise a first MP305-derived chromosomal interval defined by BNLG2162 and UMC1051, and not by a second MP305-derived chromosomal interval above UMC2041 or below UMC1051, and where the corn seeds comprise at least one of the Rcgl and Rcglb genes and, when grown, produce a corn plant that exhibits resistance to Colletotrichum infection. Seeds of embodiments also include corn seeds comprising a first chromosomal interval derived from MP305 but not including UMC2285 and UMC15a, and not including a second chromosomal interval derived from MP305 at or above UMC2285 or below UMC15a, and in addition to corn seeds such as those comprising the Rcgl and Rcglb genes and when grown, produce a corn plant that exhibits resistance to Colletotrichum infection. Corn plants and plant cells produced from such seeds are also included in embodiments of the invention.

Progenic seed that is a conversion of the Rcgl locus of PH705, PH5W4, PH51K or PH87P, or a progeny thereof is also embodied in the invention, as are seeds that are progeny comprising at least two or more of MZA11123 allele 7, allele 2 of MZA2591, or allele 8 of MZA3434. Additional embodiments include progenic seeds comprising a cytosine nucleotide in MZA2591.32, a thymine nucleotide in MZA2591.35, and a cytosine nucleotide in MZA3434.17.

Embodiments also include genetic markers at or within SEQ ID NOs: 140 to 146 for MZA3434, MZA2591, MZA11123, MZA15842, MZA1851, MZA8761 and MZA11455, respectively. Other embodiments include genetic markers located on or in the Rcgl locus or the Rcgl gene or the Rcglb gene, including those located in SEQ ID NO: 137 or in any of SEQ ID NOs: 255, 256, 257, 258 and 266. Incorporated Markers also include those located in SEQ ID NO: 1 and SEQ ID NO: 245.

Brief description of the figures

Figure 1 is a map of the United States showing the severity of anthracnose stalk rot infestation by county for 2002.

Figure 2 (a, b, c) is an alignment of a polypeptide sequence of the embodiments (SEQ ID NO: 3) comparing it to other known NBS-LRR polypeptides.

Figure 3 is a graph produced by Windows QTL Cartographer software showing a statistical analysis of chance (Y-axis) that the locus responsible for the Cg resistance phenotype is located at a certain position along the chromosome (X-axis) as defined by the markers FLP.

Figure 4 is a gel electrophoresis blot of aliquots of RT-PCR reactions, which reveals the presence of a 260 bp band present in samples from both infected and uninfected resistant plants, but absent from sensitive samples. . RT-PCR fragments were obtained from 12.5 ng of total RNA from stem tissue of DE811 and DE811ASR. cDNA obtained by reverse transcription was amplified using Rcgl-specific primers and 18S rRNA primers as internal standards.

Figure 5 is a schematic diagram of the Mutagging strategy used to validate the Rcgl gene.

Figure 6 is the structure of the Rcgl gene showing the location of the four different modifier insertion sites.

Figure 7(a-b) is a series of genetic map images with improved resolution of the map of the region near the Rcgl locus. Map distances for 7(a) the map labeled "A" are in cM and relative to the IBM2 neighbors of genetic map 4. Map distances for 7(b) the map labeled "B" were developed using 184 individuals from the BC7 population, and map distances for 7(b) the map labeled "C" were developed using 1060 individuals from the BC7 population. Genetic mapping in the BC7 population increased the map resolution by more than 10-fold compared to the published map. The locations of markers shown to the right of each map are based on extrapolation from their locations on the physical map.

Figure 8 (a-b) is an image of the genetic map showing the chromosomal interval with the Rcgl locus in DE811ASR (BC3), the reduced size of the chromosomal interval with the Rcgl locus obtained in DE811ASR (BC5) and the reduced size of the chromosomal interval in lines obtained initially using DE811ASR (BC5) as a donor source. For all markers, map distances shown were reported over the publicly available IBM2 neighbor map over the Milho GDB, in addition to MZA15842, FLP27 and FLP56 for which positions were extrapolated using regression analysis against the high-resolution maps in Figure 7(b), maps B and C, using the positions of UMC2285, PHI093 and CSU166a that were common to both maps. Figures 9 (a-b). Figure 9 (a) shows the alignment of the non-collinear region of DE811ASR (BC5) relative to B73 and Mol7. The BAC sizes in Figure 9(a) are estimates. Figure 9 (b) shows a portion of the non-collinear region as set forth in SEQ ID NO: 137 in which Rcgl resides, including the repetitive regions therein, as well as exons 1 and 2 of Rcgl.

Figures 10 (a-b) show mean leaf size lesion distributions on different individual plants, 15 days after Cg inoculation in lines DE811ASR (BC5) and DE811, respectively.

Figure 11 shows a comparison of mean leaf lesion size in DE811 and DE811ASR (BC5) plants infected with Cg 7 and 15 days after inoculation.

Figure 12 shows the mean disease severity four to five weeks after Cg inoculation in stems of hybrids derived from the cross of DE811ASR (BC5) and DE811 for the indicated line.

Figure 13 shows the improvement in maturity yield after Cg inoculation in hybrids derived from crossing DE811ASR (BC5) for the indicated line when compared with the yield of hybrids derived from crossing DE811 for the indicated line.

Figure 14 shows the severity of disease at 5 different sites caused by Cg in stems of lines derived from DE811ASR (BC5) or MP305 at four to five weeks after inoculation. Differences between strains that were positive and negative for the Rcgl gene are statistically significant at a P value of less than 0.05.

Figure 15 shows disease progression in stems representing inbred lines of PH705 that are positive and negative for Rcgl.

Figure 16 shows disease progression in stems representing PH87P inbred lines that are positive and negative for Rcgl.

Figure 17 shows the severity of the disease four to five weeks after inoculation, at 5 different locations, caused by Cg in stems of hybrids derived from crossing DE811ASR (BC5) for the indicated line. Differences between strains that were positive and negative for the Rcgl gene are statistically significant, with a P value of less than 0.05, except for position 5.

Figure 18 shows disease progression in stems representing hybrids created from lines of PH4CV and PH705 that are positive and negative for Rcgl.

Figure 19 shows disease progression in stems representing hybrids created from lines of PH705 and PH87P that are positive and negative for Rcgl.

Figure 20 presents the scoring method for disease severity in corn cobs. Stems are assigned a score, called antgr75, which represents the number of internodes (up to 5, including the inoculated internode), which are more than 75% discolored. This results in a score ranging from 0 to 5, with 0 indicating less than 75% discoloration of the inoculated internode, and 5 indicating 75% or more discoloration of the first five internodes, including the inoculated internode.

Figure 21 shows a contig on the B73 physical map that is homologous to the region in which the Rcgl non-collinear region containing DE811ASR (BC5) is inserted, which demonstrates that many B73-derived bacterial artificial chromosomes (BACs) are available in the B73 region. interest from which sequence information can be obtained.

Figure 22 shows the alignment of the genetic map containing MZA and public markers with the Mol7 and B73 physical maps. Genetic map distances were developed using 1060 individuals from the BC7 mapping population. An analysis of a Mol7 BAC library also showed that the Rcgl locus is non-collinear with the corresponding region of Mol7. The locations of markers shown by dotted lineages for the B73 map are extrapolations from the Mol7 physical location map. The locations of markers shown by dotted lineages on the Mol7 map are extrapolations from the B73 physical location map.

Figure 23 shows the oligos for the Rcgl hybridization tags designed for use with INVADER™ detection system reactions.

Figure 24 shows the oligos for the Rcgl hybridization tags designed for use with TAQMAN® detection system reactions.

Figure 25 shows the results of a Northern blot obtained from approximately 1.5 mg of polyA-enriched RNA isolated from resistant and susceptible plants 0, 3, 6, 9 and 13 days post-inoculation (dpi). The membrane was probed with a randomly labeled primer from the 420 bp fragment of Rcgl. Resistant tissue is DE811ASR (BC5) and susceptible tissue is DE811.

Figure 26 shows that PCR amplification using Rcgl-specific primer pairs only amplifies in the resistant strain DE811ASR (BC5) and donor parent MP305, but not in the susceptible strain DE811, with the exception of FLP110F-R, which amplifies the coiled-coil nucleotide binding site region, which is highly conserved and therefore amplifies a region elsewhere in the genome than Rcgl in strain DE811. A 100 bp scale was used for fragment sizing.

Figure 27 shows a series of leaf sheaths from V7-9 maize plants that were inoculated with a suspension of Cg conidia following tissue wounding. These results are discussed in Example 8. In the absence of the Rcgl transgene (superior lineage), leaf sheaths of plants containing the Rcgl gene exhibit large, expanded necrotic lesions. In contrast, when the Rcgl transgene (in this case, driven by its own promoter) is present (inferior lineage), an ASR-resistant phenotype is restored and necrosis is confined/restricted to the site of wounding and fungal inoculation.

Figure 28 shows the results of the RT-PCR performed with the primer pair ETJ115 - ETJ116. This data clearly shows that the NBS-LRR gene from the BAC 191 supercontiguous clone H is expressed exclusively in the DE811ASR plants that contain the region of the MP305 generated lines, as discussed in Example 9.

Figure 29 shows the predicted domain structure for the Rcglb gene, as discussed in Example 9.

Figure 30 shows the result of a Northern blot analysis that allowed independent confirmation of Rcglb expression and provided an estimate of transcript size. Northern blots of polyA + RNA (0.7 mg) from resistant and susceptible plants (0, 6, 13 DPI) showed the presence of a 4.75 kb transcription band, present only in resistant samples that specifically hybridized with prepared probes from LRR and the kinase region of the gene, as discussed in Example 9.

Figures 31a through 31g provide an alignment of the Rcglb peptide (SEQ ID NO: 246) of embodiments with various NBS-LRR proteins from barley and rice (SEQ ID NOs: 247-251), showing regions of homology.

Figures 32a and 32b provide an alignment of amino acids 1036-1399 of SEQ ID NO: 246, which are defined separately as SEQ ID NO: 256, with several putative protein kinases (SEQ ID NOs: 252-254) identified in a search of BLAST ®P homology.

Figure 33 summarizes the leaf sheath infection data of Rcgl KO plants in the absence and presence of the native Rcgl transgene, as discussed in Example 8.

Figure 34 shows the results of leaf sheath assays of plants transformed with Construct E as described in Example 10. A clear pattern of Cg infection is visible.

Figure 35 shows the results of leaf sheath assays of plants transformed with Construct C as described in Example 10. A clear pattern of Cg infection is visible.

Figure 36 shows the results of leaf sheath assays of plants transformed with Construct D as described in Example 10. Leaves clearly show inhibition of Cg infection as a result of the simultaneous presence and expression of both the Rcgl and Rcglb.

Detailed description of the invention

Embodiments of the present invention provide compositions and methods (or processes) directed to inducing pathogen resistance, especially fungal resistance, in plants. The compositions are novel nucleotide and amino acid sequences that confer or enhance resistance to plant pathogenic fungi. Specifically, certain embodiments provide polypeptides having the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 246, and variants and fragments thereof. Isolated nucleic acid molecules, and variants and fragments thereof, comprising nucleotide sequences encoding the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 246 are further provided.

Nucleotide sequences encoding the polypeptides of SEQ ID NO: 3 are set forth in SEQ ID NOs: 1 and 4. Nucleotide sequences encoding the polypeptides of SEQ ID NO: 246 are set forth in SEQ ID NOs: 255-258, as well as 246 and 266. The genomic sequence of the Rcglb gene and its flanking regions are provided in SEQ ID NOs: 255-258, or in SEQ ID NO: 266, which are discussed in more detail in Example 9. The sequence of the cDNA encoding the polypeptide of SEQ ID NO: 246 is set forth in SEQ ID NO: 245. Plants, plant cells, seeds and microorganisms that comprise a nucleotide sequence encoding a polypeptide of embodiments are also disclosed herein.

A deposit of the nucleic acid molecule Rcgl was made on February 22, 2006, in the Agricultural Research Service (ARS) Culture Collection, housed in the Microbial Genomics and Bioprocessing Research Unit of the National Center for Agricultural Utilization Research (NCAUR), under the provisions of the Budapest Treaty. The deposit was determined with the following accession number: NRRL B-30895. NCAUR's address is 1815 N. University Street, Peoria, IL, 61604. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Processing. This deposit has been made solely as a convenience to those skilled in the art and is not an admission that a deposit is required under USC 35 § 112. The deposit will be irrevocable and without any restriction or condition of being available to the public, by issuing a patent. However, it must be understood that the existence of a deposit does not constitute a license to practice the subject matter of the invention in derogation of patent rights granted by government action.

A sample of 2,500 seeds of DE811ASR (BC5) was deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110-2209, U.S.A. on March 13, 2006 and assigned Deposit No. PTO-7434. Access to this deposit will be available during the pendency of the application to the Commissioner of Patents and Trademarks, to persons determined by the Commissioner to be entitled to his application, and to corresponding officials in foreign patent offices where this patent application is filed. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Proceedings. The deposit will be irrevocable and without any restriction or condition to be made available to the public upon issuance of a patent. However, it should be understood that the existence of the deposit does not constitute a license to practice the subject matter of the invention or methods in derogation of patent rights.

A deposit of the Rcglb nucleic acid molecule was made on June 26, 2007, in the Agricultural Research Service (ARS) Culture Collection, housed in the Microbial Genomics and Bioprocessing Research Unit of the National Center for Agricultural Utilization Research (fNCAUR), under the provisions of the Budapest Treaty. The deposit was given with the following accession number: NRRL B-50050. The address is NCAUR 1815 N. University Street, Peoria, IL, 61604. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Processing. This deposit has been made solely as a convenience to those skilled in the art and is not an admission that a deposit is required under USC 35 § 112. The deposit will be irrevocable and without any restriction or condition of being available to the public upon issuance of a patent. However, it must be understood that the existence of a deposit does not constitute a license to practice the subject matter of the invention in derogation of patent rights granted by government action.

The two full-length polypeptides of the embodiments (SEQ ID NOs: 3 and 246) show different degrees of homology with known polypeptides of the NBS-LRR family. In particular, Rcgl (SEQ ID NO: 3) shares homology with NBS-LRR proteins isolated from Oryza sativa (n2 Accession NP_910480 (SEQ ID NO: 14), NP_910482 (SEQ ID NO: 16), NP_921091 (SEQ ID NO: 17 ) and NP_910483 (SEQ ID NO: 15)) and Hordeum vulgare (Accession no. AAG37354 (SEQ ID NO: 18); Zhou et al., (2001) Plant Cell 13:337-350). Figure 2 shows an alignment of the amino acid sequence contained in SEQ ID NO: 3 with proteins from O. sativa and H. vulgare (SEQ ID NOs: 14-18). Amino acid alignments using the GAP program to compare SEQ ID NO: 3 to various rice and barley sequences identified above indicated that SEQ ID NO: 3 shares approximately 42.3% sequence similarity with protein from O. sativa NP_910480 ( SEQ ID NO: 14), 41.7% sequence similarity with O. sativa protein NP_910482 (SEQ ID NO: 16), 56.9% similarity with O. sativa protein NP_921091 (SEQ ID NO: 17) and 42.1% sequence similarity with protein from O. sativa NP_910483 (SEQ ID NO: 15). Furthermore, SEQ ID NO: 3 shares approximately 42.8% sequence similarity with the H. vulgare protein AAG37354 (SEQ ID NO:

Similar analysis was performed for the Rcglb peptide (SEQ ID NO: 246), which was found to share homology with NBS-LRR proteins isolated from Oryza sativa (Accession Nos. ABA95308 (SEQ ID NO: 250), ABG66042 (SEQ ID NO: 251 ), ABA92208 (SEQ ID NO: 247) and ABG66044 (SEQ ID NO: 248)) and Hordeum vulgare (Accession No. AAM22820 (SEQ ID NO: 249)). Figure 31a to 31g provides an alignment of the amino acid sequence set forth in SEQ ID NO: 246 with proteins from O. sativa and H. vulgare with the accession numbers mentioned above (SEQ ID NOs: 247-251). Amino acid alignments using the ClustalW pairwise alignment (Opening gap = 10, Extending gap = 0.1) indicate that SEQ ID NO: 246 shares 37.1% sequence identity with the O. sativa ABA92208 protein (SEQ ID NO: 247), 28.2% sequence identity with the O. sativa protein ABG66044 (SEQ ID NO: 248), 26.6% identity with the O. sativa protein ABA95308 (SEQ ID NO: : 250) and 31.1% sequence similarity with the O. sativa ABG66042 protein (SEQ ID NO: 251). Furthermore, SEQ ID NO: 3 shares approximately 25.6% sequence similarity with the H. vulgare AAM22820 protein (SEQ ID NO: 249). The NBS-LRR group of R-genes is the largest class of R-genes discovered so far. In Arabidopsis thaliana, more than 150 are predicted to be present in the genome (Meyers, et al., (2003), Plant Cell, 15:809-834; Monosi, et al., (2004), Theoretical and Applied Genetics, 109 :1434-1447), while in rice, about 500 NBS-LRR genes have been predicted (Monosi, (2004 supra)). The NBS-LRR gene class is composed of two subclasses. Class 1 NBS-LRR genes that contain a TIR-Toll/Interleukin-1 like domain at their N' terminus; which until now have only been found in dicotyledons (Meyers, (2003) supra; Monosi, (2004 supra)). The second class of NBS-LRR contains a coiled-coil domain or an NT (domain) at its N terminus (Bai et al. (2002) Genome Research, 12:1871 -1884; Monosi, (2004) supra; Pan, et al. , (2000), Journal of Molecular Evolution, 50:203-213). Class 2 NBS-LRR has been found in both dicot and monocot species. (Bai, (2002) supra; Meyers, (2003) supra; Monosi, (2004) supra; Pan, (2000 supra)).

The NBS domain of the gene appears to have a signaling role in plant defense mechanisms (van der Biezen, et al., (1998), "Current Biology: CB, 8: R226-R227). The LRR region appears to be the region which interacts with the AVR products of pathogens (Michelmore, et al., (1998), Genome Res., 8:1113-1130; Meyers, (2003 supra)). much greater selection pressure to diversify (Michelmore, (1998) supra; Meyers, (2003) supra; Palomino, et al., (2002), Genome Research, 12:1305-1315). thus, these 20-29-residue motifs are present in tandem arrays in a number of proteins with diverse functions, such as hormone-receptor interactions, enzyme inhibition, cell adhesion, and cell trafficking. LRR proteins in early mammalian development, neural development, cell polarization, regulation of gene expression, and apoptosis signaling. The gene of the embodiments is clearly related to the class 2 NBS-LRR family, but does not completely fit the classical mold. The terminal amino acid has homology with so-called nucleotide binding sites (NBS). There is a leucine-rich region, located, as expected, downstream of the NBS. However, unlike previously studied NBS-LRR proteins, the leucine-rich region lacks the systematic repetitive nature found in most classical LRR domains, much less consistently following the typical LXX repeat pattern and in particular, lacking instances of the consensus sequences described by Wang et al. ((1999) Plant J. 19:55-64; see especially, fig. 5), or Bryan et al. ((2000), Plant Cell 12:2033 -- 2045; see especially, fig. 3). Rcglb, in addition to the NBS and LRR domains, contains a protein kinase domain.

Because the LRR region is the receptor portion of an NBS-LRR, when a new LRR such as those of this disclosure are found, the range of its activity, that is, the number of pathogens to which it will respond, is not immediately obvious from the sequence. The modality genes were isolated from the Cg resistance phenotype and, therefore, the new LRR responds to Cg. However, it is not excluded that they respond to other pathogens not tested in the work done so far.

The nucleic acids and polypeptides of the embodiments are useful in methods of conferring or increasing fungal resistance of a plant. Thus, the compositions and methods disclosed herein are useful in protecting plants from fungal pathogens. "Pathogen resistance", "fungal resistance", and "disease resistance" are intended to say that the plant avoids disease symptoms, which are the result of plant-pathogen interactions. That is, pathogens are prevented from causing plant disease and associated disease symptoms, or alternatively, the symptoms of the disease caused by the pathogen are minimized or lessened, for example, reduced stress and associated productivity loss. One skilled in the art will appreciate that the compositions and methods disclosed herein can be used with other compositions and methods available in the art of protecting plants from pathogen attack.

Thus, the methods of the embodiments can be used to protect plants from disease, particularly diseases that are caused by plant pathogenic fungi. As used herein, "fungal resistance" refers to the greater resistance or tolerance to a fungus as compared to that of a wild-type plant. The effects can range from a slight increase in tolerance to the effects of the fungal pathogen (e.g., partial inhibition) to complete resistance such that the plant is not affected by the presence of the fungal pathogen. An increased level of resistance against a particular fungal pathogen or against a broader spectrum of fungi constitutes an "enhancement", or improved fungal resistance. Embodiments of the invention will also increase or improve the fungal pathogen resistance of plants, such that the plant's resistance to a fungal pathogen or pathogens will increase. The term "increase" refers to improving, increasing, amplifying, multiplying, elevating, raising, and so on. Here, the plants of the invention are described as being resistant to Cg infection or having "improved resistance" to Cg infection as a result of the Rcgl locus of the invention. Therefore, they typically exhibit greater disease resistance when compared to equivalent plants that are susceptible to Cg infection because they have lost the Rcgl locus. For example, using the scoring system described in Example 11 (see also figure 20), they typically display a one point, two point, or three point or more drop in infection score, or even a count reduction of 1 or 0. when compared to equivalent plants that are susceptible to Cg infection because they have lost the Rcgl locus.

In certain aspects, methods of assigning or improving fungal resistance in a plant comprise introducing into a plant at least one expression cassette, wherein the expression cassette comprises a nucleotide sequence encoding the antifungal polypeptides of each embodiment operatively. linked to a promoter that directs expression in the plant. The plant expresses both polypeptides, which confers resistance to fungi upon the plant, or improves the plant's inherent level of resistance. In certain embodiments, the gene confers resistance to the fungal pathogen, Cg. The person skilled in the art can, if desired, create two separate transgenic plants each expressing one of the two polypeptides and then crossing the two plants, selecting plants that express both polypeptides.

Expression of an antifungal polypeptide from the incorporations can be targeted to specific plant tissues where resistance to the pathogen is particularly important, such as, for example, the leaves, roots, stems, or vascular tissues of the tissue. This tissue-preferred expression can be made by root-preferred, leaf-preferred, vascular-tissue-preferred, stem-preferred, or seed-preferred promoters.

As used herein, "nucleic acid" includes a reference to a ribonucleotide or deoxyribonucleotide polymer in any single or double-stranded form, and, unless otherwise limited, encompasses known analogues (e.g., peptide nucleic acids) with the essential nature of nucleotides. in that they cross-link single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.

The terms "polypeptide", "peptide", and "proteins" are used interchangeably herein to refer to an amino acid residue polymer. The terms applicable to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding natural amino acid, as well as naturally occurring amino acid polymers. Polypeptides of the embodiments can be produced from a nucleic acid disclosed herein, or by use of standard molecular biology techniques. For example, a truncated protein from the embodiments can be produced by expressing a recombinant nucleic acid from the embodiments in an appropriate host cell, or alternatively by a combination of ex vivo procedures, such as protease digestion and purification.

As used herein, the terms "encoding" or "encoded" when used in the context of a particular nucleic acid means that the nucleic acid comprises the information necessary to directly translate the nucleotide sequence into a specific protein. The information by which a protein is encoded is specified by the use of codons. A nucleic acid encoding a protein may include untranslated sequences (e.g., introns) in translated regions of the nucleic acid or may lose such intervening, untranslated sequences (e.g., as in DNA).

Embodiments of the invention encompass isolated or substantially purified polynucleotides or protein compositions. An "isolated" or "purified" polynucleotide or protein, or biologically active parts thereof, is substantially or essentially free of components that normally accompany or interact with the polynucleotides or proteins as found in their natural environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other genetic material, or culture medium, when produced by recombination techniques (i.e. PCR amplification), or substantially free of chemical precursors or other chemicals when chemically synthesized. Ideally, an "isolated" polynucleotide is free of sequences (e.g., protein-coding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the DNA of the organism's genome from from which the polynucleotide is derived. For example, in various embodiments, the isolated polynucleotides may contain less than about 5 kb, about 4 kb, about 3 kb, about 2 kb, about 1 kb, about 0.5 kb, or about 0. .1 kb of nucleotide sequence that naturally flanks the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of genetic material includes the preparation of proteins with less than about 30%, about 20%, about 10%, about 5%, or about 1% (by dry weight) contaminating proteins. . When the protein of the embodiment, or a biologically active portion thereof, is recombinantly produced, the optimal culture medium represents less than about 30%, about 20%, about 10%, about 5%, or about 1%. (in dry weight) of chemical precursors or non-protein chemicals of interest.

Disclosed fragments and variants of nucleotide sequences and encoded proteins are also covered by the modality. By "Fragment" is meant a portion of the nucleotide sequence or a part of the amino acid sequence and, consequently, of the protein thus encoded. Fragments of a nucleotide sequence can encode protein fragments that retain the biological activity of the native protein and therefore have the ability to confer fungal resistance in a plant. Alternatively, fragments of a nucleotide sequence that are useful as hybridization probes do not necessarily encode protein fragments retaining biological activity. Thus, fragments of a nucleotide sequence can range from at least about 15 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence encoding the polypeptides of the embodiments.

A fragment of a nucleotide sequence encoding a biologically active portion of a polypeptide of the embodiments will encode at least about 15, about 25, about 30, about 40, or about 50 contiguous amino acids, or up to the total number of amino acids present in a full-length polypeptide of the embodiments (e.g., 980 amino acids for the peptide encoded by SEQ ID NO: 1). Fragments of a nucleotide sequence that are useful as hybridization probes or PCR primers generally do not need to encode a biologically active portion of a protein.

As used herein "full-length sequence", in reference to a specified polynucleotide, means having the entire nucleic acid sequence of the native sequence. "Native sequence" means an endogenous sequence, i.e., a non-engineered sequence found in the genome of an organism.

Thus, a fragment of a nucleotide sequence of embodiments may encode a biologically active portion of a polypeptide, or may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. A biologically active portion of an antipathogenic polypeptide can be obtained by isolating a portion of one of the nucleotide sequences of the embodiments, expressing the encoded portion of the protein, and evaluating the ability of the encoded portion of the protein to confer or enhance resistance to fungi in a plant. Nucleic acid molecules that are fragments of a nucleotide sequence of embodiments include at least about 15, about 20, about 50, about 75, about 100, or about 150 nucleotides, or up to the number of nucleotides present in a full-length nucleotide sequence disclosed herein (e.g., 4212 nucleotides for SEQ ID NO: 1).

"Variants" are intended for substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and/or substitution of one or more nucleotides at one or more sites within the native polynucleotide. As used herein, a native "polynucleotide" or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. One skilled in the art will recognize that the nucleic acid variations of the embodiments will be constructed in such a way that the open reading frame is maintained. For polynucleotides, conservative variants include sequences that, because of degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of the embodiments. Naturally occurring allelic variants such as these can be identified using known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques, as described below. Polynucleotide variants also include synthetically derived polynucleotides, such as those generated, for example, using site-directed mutagenesis, but which still encode a protein of the embodiments. Generally, variants of a particular polynucleotide of the embodiments will be at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity for the particular polynucleotide, as determined by sequence alignment programs and parameters described herein.

Variants of a particular polynucleotide of the embodiments (i.e., the reference polynucleotide) can also be evaluated by comparing the percentage of sequence identity between the polypeptides encoded by a polynucleotide variant and the polypeptide encoded by the reference polynucleotide. Thus, for example, isolated polynucleotides encoding a polypeptide with a percentage of sequence identity given to the polypeptide of SEQ ID NOs: 3 or 246 are disclosed. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described here. When a given pair of polynucleotide embodiments is evaluated by comparing the percentage of sequence identity shared by the two polypeptides they encode, the percentage of sequence identity between the two encoded polypeptides is at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity.

"Variant" protein is a protein derived from the native protein by deleting or adding one or more amino acids at one or more internal sites in the native protein and/or substituting one or more amino acids at one or more sites in the native protein. Variant proteins integrated by the incorporations are biologically active, that is, they continue to possess the desired biological activity of the native protein, that is, the ability to confer or increase resistance to plant fungal pathogens as described herein. Such variants may result, for example, from genetic polymorphism or human manipulation. Biologically active variants of a native protein of the incorporations will have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75 %, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97 %, about 98%, about 99% or more sequence identity with the amino acid sequence for the native protein, as determined by the sequence alignment programs and parameters described herein. A biologically active variant of the protein embodiments may differ from the protein by only 1-15 amino acid residues, only about 1-10, as well as about 6-10, only about 5, only 4, 3, 2 or even 1 residue. of amino acids.

Embedding proteins can be altered in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions. Methods of such manipulations are generally known in the art. For example, amino acid sequence variants and antipathogenic protein fragments can be prepared by mutations in DNA. Methods of mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Academic. Sci. USA 82: 488-492; Kunkel et al. (1987) Methods in Enzymol. 154: 367-382; US Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC), incorporated herein by reference. Conservative substitutions, such as exchanging one amino acid for another with similar properties, may be ideal.

Thus, the genes and polynucleotides of the incorporations include both naturally occurring sequences as well as mutant forms. Likewise, incorporation proteins encompass both naturally occurring proteins as well as variations and modified forms thereof. These variants continue to possess the desired ability to confer or increase resistance to the fungal plant pathogen. Obviously, the mutations that will be made in the DNA encoding the variation must not place the reading sequence and optimally will not create complementary regions that could produce mRNA secondary structure. See, EP patent No. 0075444.

The deletions, insertions and substitutions of the protein sequences encompassed here are not expected to produce radical changes in the characteristics of the proteins. However, when it is difficult to predict the exact effect of the substitution, deletion or insertion before doing so, one skilled in the art will understand that the effect will be evaluated by selecting transgenic plants that have been transformed with the variant protein to ascertain the effect on the ability of the plant to resist attack by pathogenic fungi.

Variant polynucleotides and proteins also encompass sequences and proteins derived from mutagenic or recombinagenic procedures, including and not limited to procedures such as DNA shuffling. One skilled in the art could imagine modifications that would alter the number of pathogens to which the protein reacts. With such a procedure, one or more different protein-coding sequences can be manipulated to create a new protein that has the desired properties. In this way, recombinant polynucleotide libraries are generated from a population of related polynucleotide sequences comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, the coding sequence motifs of a domain of interest can be shuffled between the protein gene of the embodiments and other known protein genes to obtain a new gene that codes for a protein with an improved property of interest. , such as increased ability to confer or increase resistance to plant fungal pathogens. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Academic. Know. U.S. 91: 10747-10751; Stemmer (1994) Nature 370: 389-391; Crameri et al. (1997) Nature Biotech. 15: 436-438; Moore et al. (1997) J. Mol. Biol. 272: 336-347; Zhang et al. (1997) Proc. Natl. Academic. Know. U.S.A. 94: 4504-4509; Crameri et al. (1998) Nature 391:288-291, and US Patent Nos. 5,605,793 and 5,837,458.

The polynucleotides of the embodiments can be used to isolate corresponding sequences from other organisms, particularly other plants. In this way, methods such as PCR, hybridization and the like can be used to identify such sequences based on their sequence homology to the set of sequences contained herein. Sequences isolated on the basis of their sequence identity to the entire sequences set forth herein or variants and fragments thereof are not encompassed by the incorporations. Such sequences include sequences that are orthologous to the disclosed sequences. "Orthologous" refers to genes that are derived from a common ancestral gene and are found in different species as a result of speciation. Genes found in different species are considered orthologous when their nucleotide sequences and/or their encoded protein sequences share at least about 60%, about 70%, about 75%, about 80%, about 85%, about of 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than sequence identity. Functions of orthologs are often highly conserved across species. Thus, isolated polynucleotides that encode a protein that confers or increases resistance to the plant fungal pathogen and that hybridizes under severe conditions to sequences disclosed herein, or variants or fragments thereof, are not covered by the incorporations.

In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also Innis et al., Eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York) and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known PCR methods include, and are not limited to, methods using paired primers, nested primers, single specific primer, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and so on. 5

In hybridization techniques, all or part of a known polynucleotide is used as a probe that selectively hybridizes to other corresponding polynucleotides present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA) of a chosen organism. Hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments or other oligonucleotides, and may be labeled with a detectable group such as 32P, or any other detectable marker. Thus, for example, hybridization probes can be made by synthetic oligonucleotides labeled based on the polynucleotides of the incorporations. Methods for preparing probes for hybridization and for constructing genomic and cDNA libraries are generally known in the art and are disclosed in Sambrook et al. (1989) above.

For example, an entire polynucleotide disclosed herein, or one or more parts thereof, can be used as a probe capable of specifically hybridizing to corresponding polynucleotides and messenger RNAs. To perform specific hybridization under a variety of conditions, such probes include sequences that are unique and are excellently at least about 10 nucleotides in length, at least about 15 nucleotides in length, or at least about 20 nucleotides in length. Such probes can be used to amplify corresponding polynucleotides from a chosen organism by PCR. This technique can be used to isolate additional coding sequences from a desired organism or as a diagnostic test to determine the presence of coding sequences in an organism. Hybridization techniques include hybridization and screening of plated DNA libraries (or plaques or colonies; see, for example, Sambrook et al. (1989) supra.

Hybridization of such sequences can be performed under harsh conditions. By "stringent conditions" or "harsh hybridization conditions" is meant those conditions under which a probe will hybridize to its target sequence to a greater detectable degree than other sequences (e.g., at least 2-fold). Severe conditions are sequence dependent and will be different in different circumstances. By controlling hybridization stringency and/or washing conditions, target groups that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringent conditions can be adjusted to allow for some mismatches in the sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, ideally less than 500 nucleotides in length.

Typically, the restrictive conditions will be those in which the salt concentration is less than about 1.5 M Na ions, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7 .0 to 8.3 and the temperature is at least about 30° C for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C for long probes (e.g., greater than 50 nucleotides) . Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C, and a wash in 0.IX to 2X SSC (20X SSC = 3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary conditions of moderate severity include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% of SDS at 37° C, and a wash in 0.5X to IX SSC at 55 to 60° C. Exemplary high-demand conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C, and a final wash in 0.IX SSC at 60 to 65° C for at least 30 minutes. Optionally, wash buffers may contain about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, typically about 4 to about 12 hours. The duration of the washing time will be at least a sufficient period of time to achieve equilibrium.

Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the thermal melting point (Tm) can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: T m = 81.5 ° C + 16.6 (log M) + 0.41 (%GC) - 0.61 (%form) - 500 / L, where M is the molarity of cations monovalents, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, %form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly paired probe. T m is reduced by about 10 C for each 1% match, thus T m, hybridization and/or washing conditions can be adjusted to hybridize sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tm can be reduced to 10°C. In general, stringent conditions are selected to be about 5°C lower than the Tm for the specific sequence. and its complement at the defined ionic strength and pH. However, extremely stringent conditions may utilize hybridization and/or washing at, 2, 3, or 4°C lower than the Tm; moderately harsh conditions may utilize hybridization and/or washing at 6, 7, 8, 9 or 10°C lower than the Tm; Low stringency conditions may utilize hybridization and/or washing at 11, 12, 13, 14, 15 or 20°C lower than the Tm. Using the equation, hybridization and washing compositions, and desired Tm, those skilled in the art will understand that variations in the severity of hybridization and/or washing solutions are inherently described. If the desired degree of matching results at a Tm of less than 45°C (aqueous solution) or 32°C (formamide solution), it is ideal to increase the concentration of the CCD so that a higher temperature can be used. An extensive guide to nucleic acid hybridization is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York) and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) above.

Various procedures can be used to verify the presence or absence of a particular DNA, RNA, or protein sequence. These include, for example, Southern blots, northern blots, western blots, and ELISA analysis. Techniques such as these are well known to those skilled in the art and there are many references that provide detailed protocols. Such references include Sambrook et al. supra (1989), and Crowther, JR (2001), The ELISA Guidebook, Humana Press, Totowa, NJ, USA.

The following terms are used to describe the sequence relationships of two or more polynucleotides or polypeptides: (a) "reference sequence" (b) "comparison window", (c) "sequence identity", and, (d) "percent sequence identity". (a) As used herein, "reference sequence" is a defined sequence used as a basis of sequence comparison. A reference sequence may be a subset or the entirety of a specific sequence, for example, as a full-length cDNA segment or gene sequence, or complete cDNA or gene sequence. (b) As used herein, "comparison window" refers to a precise, contiguous segment of a nucleotide sequence, wherein the nucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) relative to the reference sequence (which does not include additions or deletions) for the best alignment of the two polynucleotides. Generally, the comparison window is at least about 20 contiguous nucleotides in length, and optionally may be about 30, about 40, about 50, about 100, or more. Those skilled in the art understand that, to avoid high similarity to a reference sequence, due to the inclusion of gaps in the polynucleotide sequence an opening gap is generally introduced and is subtracted from the number of matches.

Sequence alignment methods for comparison are well known in the art. Thus, determining the percentage of sequence identity between the two sequences can be done using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, the search local alignment method of Pearson and Lipman (1988) Proc. Natl. Academic. Sci.. 85:2444-2448, the algorithm of Karlin and Altschul (1990) Proc. Natl. Academic. Sci. USA 872264, with changes from Karlin and Altschul (1993) Proc. Natl. Academic. Sci. USA 90:5873-5877.

Computerized implementations of these mathematical algorithms can be used to compare sequences to determine sequence identity. Such implementations include, and are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California), the ALIGN program (Version 2.0), and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Software Package GCG Wisconsin Genetic, version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237-244 (1988), Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS 8:155-65; and Pearson et al. (1994) Method. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) above. The PAM120 residue weight table, a gap length of 12, and a gap length of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul et al (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, Average = 100, length = 12, to obtain the nucleotide sequences homologous to a nucleotide sequence encoding a protein of the embodiments. BLAST protein searches can be performed with the BLASTX program, average = 50, length = 3, to obtain amino acid sequences homologous to a protein or polypeptide of the embodiments. To obtain gapped alignments for comparison purposes, BLAST GAPPED (in BLAST 2.0) can be used as described in Altschul et al. (1997) Nucleic Acids Res 25:3389. Alternatively, PSI-BLAST (BLAST 2.0) can be used to perform a repeated search that detects distant relationships between molecules. See Altschul et al. (1997) above. When using BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g. BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See ncbi.nlm.nih.gov. Alignment can also be performed manually by inspection.

Unless otherwise stated, sequence identity/similarity values contained in this document refer to values obtained using GAP version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using Gap weight of 50 and Weight of Length of 3, and the Scoring Matrix nwsgapdna.cmp; % identity and % similarity for an amino acid sequence using Weight of

Gap of 8 and Length Weight of 2, and the BLOSUM62 Scoring Matrix, or any other equivalent program. The "equivalent" program is intended for any sequence comparison program that, for any two sequences in question, generates an alignment with identical nucleotides or amino acid residues found and a percentage of identical sequence identity when compared with the corresponding alignment generated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, to find the alignment of two complete sequences that maximize the number of matches and minimize the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of paired bases and the fewest gaps. This allows for a gap creation disadvantage and a gap extension disadvantage to be available on combined base units. GAP must generate a benefit of numbers of combinations from the disadvantage of creating the gap for each gap entered. If a gap length that generates a disadvantage greater than zero is chosen, GAP must, in addition, generate a benefit for each inserted gap of the length of the gap length disadvantage gap times. The missing gap creation disadvantageous values and gap extension disadvantageous values in version 10 of the Wisconsin Genetics GCG Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the pattern of gap creation is 50, while the pattern of difference in length is 3. Gap creation and gap length can be expressed as an integer selected from the group composed of integers from 0 to 200 So, for example, gap creation and gap extension could be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or higher.

GAP presents a member of the family of best lineups. There may be many members of this family, and no other member has a better quality. GAP shows four figures of merit for alignments: Quality, Relationship, Identity, and Similarity. Quality is the metric maximized in order to align sequences. Ratio is the quality, divided by the number of bases in the short segment. Percent identity is the percentage of symbols that actually match. Similarity percentage is the percentage of symbols that are similar. Symbols that are in front of gaps are ignored. Similarity is marked when the Score Value Matrix for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in version 10 of the Wisconsin Genetics GCG Software Package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. U.S.A. 89:10915). (c) As used herein, "sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage sequence identity is used in reference to proteins, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acid residues are replaced by other amino acid residues with similar chemical properties. (e.g. cost or hydrophobicity) and therefore do not alter the functional properties of the molecule. When sequences differ in conservative substitutions, the percentage of sequence identity can be adjusted to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Methods for making this adjustment are well known to those skilled in the art. Typically, this involves marking a conservative substitution as a partial rather than a complete mismatch, thus increasing the percentage of sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The score for conservative substitutions is calculated, by example, as implemented in the PC/GENE program (Intelligenetics, Mountain View, California). (d) As used herein, "percent sequence identity" means the value determined by comparing two optimally aligned sequences with a comparison window, wherein the portion of the nucleotide sequence in the comparison window may include additions or deletions (or i.e. gaps) as compared to the reference sequence (which does not include additions or deletions) for the best alignment of the two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid base or amino acid residues occur in both sequences to produce the number of paired positions, then dividing the number of paired positions by the total number of positions in the comparison window , and multiplying the result by 100 to generate the percentage of sequence identity.

The use of the term "polynucleotide" is not intended to limit embodiments of polynucleotides comprising DNA. Those skilled in the art will recognize that polynucleotides may include ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the embodiments also encompass all forms of sequences, including and not limited to, single-stranded forms, double-stranded forms, and the like.

Polynucleotides isolated from the embodiments can be incorporated into recombinant DNA constructs capable of introduction and replication in a host cell. A "vector" can be as a construct that includes a replication system and sequences that are capable of transcription and translation of a polypeptide coding sequence in a given host cell. Various vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, for example, Pouwels et al. Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Flevin et al. Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker. Such plant expression vectors may also contain a regulatory promoter region (e.g., a regulatory region for inducible or constitutive control, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site , a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.

The terms "recombinant construct", "expression cassette", "expression construct", "chimeric construct", "construct", "recombinant DNA construct" and "recombinant DNA fragment" are used interchangeably herein and are acid fragments nucleic. A recombinant construct comprises an artificial combination of nucleic acid fragments, including, but not limited to, regulatory sequences and coding sequences that are not found together in nature. For example, a recombinant DNA construct may include regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source and arranged in a manner different from that found in nature. This construct can be used alone or can be used in conjunction with a vector. If a vector is used then the choice of vector is dependent on the method that will be used to transform host cells, as is well known to those skilled in the art. For example, a plasmid vector can be used. The person skilled in the art is well aware of the genetic elements that must be present in the vector in order to be able to transform, select and propagate host cells comprising any of the nucleic acid fragments isolated from the embodiments. Screening to obtain strains exhibiting the desired expression level and pattern of polynucleotides or the Rcgl locus can be achieved by amplification, Southern analysis of DNA, Northern analysis of mRNA expression, immunoblot analysis of protein expression, phenotypic analysis, and so on. onwards. The term "recombinant DNA construct" refers to a DNA construct assembled from nucleic acid fragments obtained from different sources. The types and origins of nucleic acid fragments can be very diverse.

In some embodiments, expression cassettes containing a promoter operably linked to a heterologous nucleotide sequence of the embodiments are further provided. The expression cassettes of the embodiments find use in generating transformed plants, plant cells and microorganisms and in practicing methods for inducing resistance to fungal pathogens disclosed herein. The expression cassette will include 5' and 3' regulatory sequences operably linked to a polynucleotide of the embodiments. "Operationally linked" is intended to mean a functional connection between two or more elements. "Regulatory sequences" refer to nucleotides located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which can influence transcription, processing, RNA, stability, or translation of the associated coding sequence. Regulatory sequences may include, and are not limited to, promoters, leader translation sequences, introns, and polyadenylation recognition sequences. For example, an operational link between a polynucleotide of interest and a regulatory sequence (a promoter, for example) is a functional link that allows expression of the polynucleotide of interest. Operationally linked elements can be contiguous or non-contiguous. When used to refer to the union of two protein-coding regions, by operably linked it is meant that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional gene to be co-transformed in the organism. Alternatively, additional gene(s) may be provided in multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the polynucleotide encoding an antipathogenic polypeptide that is under the transcriptional regulation of the regulatory regions. The expression cassette may further contain selectable marker genes.

The expression cassette will include in the 5'-3' direction of transcription, a transcription initiation region (i.e., a promoter), translation initiation region, a polynucleotide of the embodiments, a translation termination region and, optionally, a functional transcription termination region in the host organism. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translation termination regions) and/or the polynucleotide of the embodiments may be native/analogous to those of the host cell or to each other. Alternatively, the regulatory regions and/or polynucleotide of the embodiments may be heterologous to the host cell or to each other. As used herein, "heterologous" in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from the native form in its composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species other than the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from its original form and/or genomic locus, or the promoter is not the native promoter of the operably linked polynucleotide.

The optionally included termination region may be native to the transcription initiation region, may be native to the operably linked polynucleotide of interest, may be native to the host plant, or may be derived from another source (i.e., foreign or heterologous) to the promoter, the polynucleotide of interest, the host, or any combination thereof. Convenient termination regions are available from the A. tumefaciens Ti plasmid, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141- 144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261 - 1272; Munroe et al. (1990) Gene 91:151-158; Bailas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639. In particular embodiments, the potato protease terminator inhibitor II (PinII) gene is used. See, for example, Keil et al. (1986) Nucl. Acids Res. 14:5641- 5650; and An et al. (1989) Plant Cell 1:115-122, incorporated herein by reference in their entireties.

A number of promoters may be used in practicing the embodiments, including native promoters of the polynucleotide sequences of interest. Promoters may be selected based on the desired outcome. A wide range of plant promoters are discussed in the recent review by Potenza et al. (2004) In Vitro Cell Dev Biol - Plant 40:1-22, incorporated herein by reference. For example, nucleic acids may be combined with constitutive, tissue-preferred, pathogen-inducible, or other promoters for expression in plants. Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and US Patent No. 6,072,050; the CaMV 35S core promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); BUT (Velten et al. (1984) EMBO J. 3:2723 - 2730); ALS promoter (US Patent No. 5,659,026), and so on. Other constitutive promoters are included, for example, in US Patents 5,608,149, 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463, 5,608,142 and 6,177,611.

It can sometimes be beneficial to express genes from an inducible promoter, particularly from a pathogen-inducible promoter. Such promoters include those of pathogen-related proteins (PR proteins), which are induced following infection by a pathogen, e.g., PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Net. J. Plant Pathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116. See also WO 99/43819, incorporated herein by reference.

Of interest are promoters that result in the expression of a protein at or near the site of pathogen infection. See, for example, Marineau et al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al. (1986) Proc. Natl. Academic. Sci. USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Academic. Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J. 10:955-966, Zhang et al. (1994) Proc. Natl. Academic. Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968; US Patent No. 5,750,386 (nematode-inducible) and references cited therein. Of particular interest is the inducible promoter for the maize PRms gene, whose expression is induced by the fungus Fusarium moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).

Furthermore, as pathogens find entry into plants through wounds or insect damage, a wound-inducible promoter can be used in the constructions of the embodiments. Such wound-inducible promoters include potato proteinase inhibitor genes PIN (II) (Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology 14:494-498); wunl and wun2, US Patent No. 5,428,148; winl and win2 (Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl et al. (1992) Science 225:1570-1573); wipl (Rohmeier et al. (1993) Plant Mol. Biol. 22:783-792, RH et al. (1993) FEES Letters 323:73-76); MPI gene (Corderok et al. (1994) Plant J. 6 (2):141-150); and the like, incorporated herein by reference.

Chemically regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending on the objective, the promoter can be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressive promoter, where application of the chemical represses gene expression. Chemically inducible promoters are known in the art and include, and are not limited to, the corn In2-2 promoter, which is activated by benzenesulfonamide herbicide protectants, the corn GST promoter, which is activated by electrophilic hydrophobic compounds that are used as pre-emergent herbicides, and the tobacco PR-Ia promoter, which is activated by salicylic acid. Other chemically regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. U.S. 88:10421-10425 and McNellis et al. ( 1998) Plant J. 14 (2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and Patents US Nos. 5,814,618 and 5,789,156), incorporated herein by reference.

Tissue-preferred promoters can be used to achieve greater expression of the polypeptides of the embodiments within a particular plant tissue. For example, a tissue-preferred promoter can be used to express a polypeptide in a plant tissue where disease resistance is particularly important, such as, for example, the roots, stem or leaves. Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12 (2): 255-265; Kawamata et al. (1997) Plant Cell Physiol. 38 (7): 792-803, Hansen et al. (1997) Mol. Gen Genet. 254 (3):337-343; Russell et al. (1997) Transgenic Res. 6 (2):157-168; Rinehart et al. (1996) Plant Physiol. 112 (3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112 (2):525-535; Canevascini et al. (1996) Plant Physiol. 112 (2): 513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35 (5):773-778; Lam (1994) Results Probl. Cell Difference. 20:181-196; Orozco et al. (1993) Plant Mol Biol. 23 (6): 1129-1138; Matsuoka et al. (1993) Proc Natl. Academic. Sci. USA 90 (20): 9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4 (3): 495-505. Such promoters can be modified, if necessary, for poor expression.

Vascular tissue-preferred promoters are known in the art and include promoters that selectively direct protein expression, for example, in the xylem and phloem. Preferred vascular-tissue promoters include, and are not limited to, the Prunus serotina prunasin hydrolase gene promoter (see, for example, International Publication No. WO 03/006651), and also those found in Patent Application Serial No. 10/ 109, 488.

Stem-preferred promoters can be used to direct expression of a polypeptide of the embodiments. Exemplary stem-preferred promoters include the maize-15 MS8 gene promoter (see, for example, US Patent No. 5,986,174 and International Publication No. WO 98/00533), and those found in Graham et al. (1997) Plant Mol Biol 33 (4): 729-735.

Preferred sheet promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12 (2):255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35 (5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23 (6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Academic. Sci. USA 90 (20):9586-9590.

Root-preferred promoters are known and can be selected from the many available in the literature or isolated de novo from several compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol. 20 (2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3 (10):1051-1061 (root-specific control element in French bean GRP 1.8 gene); Sanger et al. (1990) Plant Mol. Biol. 14 (3): 433-443 (root-specific promoter of the mannopine synthase (MAS) gene from Agrobacterium tumefaciens) and Miao et al. (1991) Plant Cell 3 (1):11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in soybean roots and root nodules). See also Bogusz et al. (1990) Plant Cell 2 (7):633-641, where two root-specific promoters isolated from hemoglobin genes of the non-nitrogen-fixing legume Parasponia andersonii and the non-nitrogen-fixing non-legume Trema tomentosa are described. The promoters of these genes were linked to the β-glucuronidase reporter gene and introduced into both the non-legume Nicotiana tabacum and the legume Lotus corniculatus, and in both cases, the activity of the root-specific promoter was preserved. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed root-inducible rolC and rold genes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79 (1):69-76). They concluded that DNA enhancer and tissue-preferred determinants are dissociated at promoters. Teeri et al. (1989) used the lacZ gene fusion to show that the Agrobacterium T-DNA gene encoding the octopine synthase enzyme is especially active in the epidermis of the root tip and that the TR2 gene is root specific in the intact plant and stimulated by tissue wounding. foliar, an especially desirable combination of traits for use with an insecticidal or larvicide gene (see EMBO gene J. 8(2):343-350). The TR1' gene, fused to nptll (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VÍENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. 29 (4):759-772); and rolb promoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See also U.S. Patent Nos. 5,837,876, 5,750,386, 5,633,363, 5,459,252, 5,401,836 , 5,110,732 and 5,023,179.

"Seed-preferred" promoters include both "seed-specific" promoters (those promoters active during seed development, such as seed storage protein promoters) as well as "germinating seed" promoters (those promoters active during seed development). seed germination). See Thompson et al. (1989) BioEssays 10:108, incorporated herein by reference. Such preferred seed promoters include and are not limited to, Ciml (message-induced cytokinin); cZ19Bl (19 kDa corn zein); milps (myo-inositol-1-phosphate synthase) (see WO 00/11177 and US Patent No. 6,225,529; incorporated herein by reference). Gamma-Zein is an endosperm-specific promoter. Glob-1 is an embryo-specific promoter. For dicots, seed-specific promoters include and are not limited to, bean β-phaseolin, napin, β-conglycinin, soy lecithin, cruciferin, and so on. For monocots, seed-specific promoters include and are not limited to, 15 kDa corn zein, 22 kDa zein, 27 kDa zein, g-Zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO 00/12733, where seed-preferred promoters of endl and end2 genes are reported; incorporated herein by reference.

Additional sequence modifications are known to increase gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron linkage site signals, transposon-like repeats, and other well-characterized sequences that may be detrimental to gene expression. The G-C content of the sequence can be adjusted to average levels for a given cellular host, calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid secondary loop structures of the mRNA.

Expression cassettes may also contain 5' leader sequences. Such leader sequences may act to improve translation. Translation leaders are known in the art and include: picornavirus leaders, e.g. EMCV leader (Encephalomyocarditis 5' non-coding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. U.S.A. 86:6126 -6130); potivirus leaders, e.g., TEV (Tobacco Etch Virus) leader (Gallie et al. (1995) Gene 165(2):233-238), MDMV (Maize Dwarf Mosaic Virus) leader, and human immunoglobulin heavy chain binding protein (BIP) (Macejak et al. (1991) Nature 353:90-94); untranslated leader of alfalfa mosaic virus capsid protein mRNA (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625), leader of tobacco mosaic virus (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256) and leader of maize chlorotic mottle virus (MCMV) (Lommel et al. (1991) Virology 81:382- 385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968. Other known methods for improving translation can also be used, for example, introns, and so on.

In the construction of the expression cassette, the various DNA fragments may be manipulated so as to provide the DNA sequences in the correct orientation and, where appropriate, in the correct reading frame. To this end, adaptors or linkers may be used to join the DNA fragments, or other manipulations may be involved to provide convenient restriction sites, remove superfluous DNA, remove restriction sites, or the like. To this end, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

The expression cassette may also include a selectable marker gene for selection of transformed cells. Selectable marker genes are used for selection of transformed cells or tissues. Marker genes include genes that encode antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes that confer resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan fluorescent protein (CYP) (Boite et al. (2004) J. Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), and yellow fluorescent protein (PhiYFP™ from Evrogen, see, Boite et al. (2004) J. Cell Science 117:943- 54). For additional selectable markers, see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl. Academic. Know. U.S. 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) No operon, pp. 177-220; Hu et al. (1987) Cell 48:555-566, Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Academic. ACI. U.S. 86:5400-5404; Fuerst et .al.. - (1989) Proc. Natl. Academic. Know. U.S. 86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl. Academic. Know. U.S.A. 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc. Natl. Academic. Know. U.S. 89:3952-3956; Baim et al. (1991) Proc. Natl. Academic. USA 88:5072-507 6; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb et al. (1991) Antimicrob. AgentsChemother. 35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc. Natl. Academic. Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology, vol. 78 (Springer-Verlag, Berlin), Gill et al. (1988) Nature 334:721-724. Such disclosures are incorporated herein by reference.

The above list of selectable marker genes is not intended to be a limitation. Any selection marker gene can be used in the embodiments.

The embodiment genes can be expressed as a transgene to make plants resistant to Cg. Using different promoters described in this disclosure will allow their expression in a modulated manner under different circumstances. For example, higher levels of expression in stems may be desired to improve resistance to Cg stem rot. In environments where Cg leaf blight is more of a problem, lines with higher levels of expression in leaves may be used. However, it is also possible to insert the entire genes, both the native promoter and the coding sequence, as a transgene. Finally, using the embodiment genes as transgenes will allow for rapid combination with other traits, such as insect or herbicide resistance.

In certain embodiments the nucleic acid sequences of the embodiments can be stacked with any combination of nucleotide sequences of interest in order to create plants with a desired phenotype. This stacking can be achieved by a combination of genes within the DNA construct, or by a passage of a lineage containing Rcgl and Rcglb (as transgenes or as a generated locus), with another lineage comprising the combination. For example, the polynucleotides of the embodiments may be stacked with any polynucleotides of other embodiments, or with other genes. The generated combinations may also include multiple copies of any of the polynucleotides of interest. The polynucleotides of the embodiments may also be stacked with any other gene or a combination of genes. to produce plants with a variety of desired characteristic combinations including and not limited to traits desirable for animal feed, such as superior oil genes (e.g., US Patent No. 6,232,529); .389, 5,885,801, 5,885,802 and 5,703,409); barley higher lysine (Williamson et al. (1987) Eur. J. Biochem. 165:99-106 and WO 98/20122) and higher methionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359; and Musumura et al. (1989) Plant: Mol. Biol. 12: 123)); increased digestibility (e.g., modified storage proteins (US Application No. 10/053, 410, filed November 7, 2001) and thioredoxins (US Application No. 10/005, 429, filed December 3, 2001 )), the disclosures of which are incorporated herein by reference. The polynucleotides of the embodiments may also be stacked with desirable characteristics for insect, disease, or herbicide resistance (e.g., toxic proteins from Bacillus thuringiensis (US Patent 5,366,892; 5,747,450, 5,737,514; 5,723,756; 5,593,881; Geiser et al (1986) Gene 48:109); lectins (Van Damme et al. (1994) Plant Mol. Biol. 24: 825); disease resistance (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; Mindrinos et al. (1994) Cell 78:1089); herbicide resistance such as the S4 and/or Hra mutations; glutamine synthetase inhibitors such as phosphinothricin or Basta (e.g. bar gene) and glyphosate resistance (EPSPS genes, GAT genes such as those disclosed in Patent Application Publication US2004/0082770 , also WO02/36782 and W003/092360)); and desirable characteristics for processing and transforming products such as superior oil (e.g., US Patent No. 6,232,529); modified oils (e.g., fatty acid desaturase genes (US Patent No. 5,952,544; WO 94/11516)); modified starches (e.g. ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (SDBE)); and polymers or bioplastics (e.g., US Patent No. 5,602,321; beta-synthase, polyhydroxybutyrate ketothiolase, and acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol. 170: 5837-5847) to facilitate the expression of polyhydroxyalkanoates (PHAs)), the disclosures of which are incorporated herein by reference. We could also combine the polynucleotides of the embodiments with polynucleotides providing agronomic characteristics, such as male sterility (e.g., see US Patent 5,583,210), stem strength, flowering time, or transformation technology characteristics, such as regulation of cell cycle or target gene (e.g. WO 99/61619, WO 00/17364, WO 99/25821), the disclosures of which are incorporated herein by reference.

These stacked combinations can be created by any method, including and not limited to crossing breeding plants by any conventional or topcross® methodology or genetic transformation. If traits are stacked by genetically transforming plants, the nucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant that comprises one or more desired traits can be used as a target to introduce new traits for further transformation. Traces can be introduced simultaneously into a co-transformation protocol with the polynucleotides of interest, by any combination of transformation cassettes. For example, if two sequences are to be introduced, the two sequences may be contained in separate transformation cassettes (trans) or contained in the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress expression of the polynucleotide of interest. This can be combined with any combination of suppression cassettes or other overexpression cassettes to generate the desired combination of traits in the plant.

As shown in the examples, the Rcgl locus contains two components, Rcgl and Rcglb, which when expressed together provide the phenotype of the present invention. One skilled in the art will recognize that if genes are being used transgenically, this could be done in a single chimeric construct inserted into a plant cell to make a transgenic plant, but it could also be done by inserting two genes as separate chimeric constructs. in plant cells to make two strains of transgenic plants. These could be crossed and progeny lines carrying both genes selected using the methods described herein to obtain pathogen-resistant lines. If one finds a strain in nature that expresses only one or the other gene, a chimeric construct containing only the missing gene can be used to make a transgenic strain expressing both genes.

The methods of the embodiments may involve, and are not limited to, introducing a polypeptide or polynucleotide into a plant. "Introducing" is intended to mean introducing the polynucleotide into the plant. In some embodiments, the polynucleotide will be introduced such that the sequence gains access to the interior of a plant cell, including its potential for insertion into the genome of a plant. The methods of the embodiments do not rely on a specific method for introducing a sequence into a plant, only that the polynucleotide gains access to the interior of at least one plant cell. Methods of introducing polynucleotides into plants are known in the art, including, but not limited to, stable transformation methods, transient transformation methods, and viral-mediated methods. "Transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms. "Host cell" refers to the cell in which transformation of the recombinant DNA construct occurs and may include a yeast cell, a bacterial cell, and a plant cell. Examples of plant transformation methods include Agrobacterium-mediated transformation (De Blaere et al. 1987, Meth. Enzymol. 143:277) and accelerated particle, or "biolistic" transformation technology (Klein et al., 1987, Nature (London) 327:70-73; U.S. Patent No. 4,945,050), among others. "Stable transformation" is intended to mean that the nucleotide construct introduced into a plant integrates into the plant genome and is capable of being inherited by the plant's descendants. "Transient transformation" or "transient expression" is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the plant genome or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e. monocot or dicot, oriented towards transformation. Suitable methods for introducing polypeptides and polynucleotides into plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. U.S. 83:5602-5606, Agrobacterium-mediated transformation (US patents 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, e.g., Sanford et al., US Patents 4,945,050, 5,879,918, 5,886,244 and 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. -Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926) and Led transformation (WO 00/28058). -477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); / Technology 6:923-926 (soy); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soy); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soy); Datta et al. (1990) Biotechnology 8:736-740 (rice), Klein et al. (1988) Proc. Natl. Academic. Sci. USA 85:4305-4309 (corn), Klein et al. (1988) Biotechnology 6:559-563 (corn); In US Patents 5,240,855, 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol. 91:440-444 (corn); Fromm et al. Biotechnology (1990) 8:833-839 (corn); Hooykaas Slogteren-Van et al. (1984) Nature (London) 311:763-764; US Patent No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. . Academic. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (Swiss mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation), Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (corn via Agrobacterium tumefaciens), which are incorporated herein by reference.

Methods are known in the art for the targeted insertion of a polynucleotide into a specific location in the plant genome. In one embodiment, insertion of the polynucleotide into a desired genomic location is achieved using a site-specific recombination system. See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855 and WO99/25853, all of which are incorporated herein by reference. Briefly, the polynucleotide of the incorporations can be contained in a transfer cassette flanked by two non-identical recombination sites. The transfer cassette is introduced into a plant having stably incorporated into its destination site a genome that is flanked by two non-identical recombination sites that correspond to the transfer cassette sites. A suitable recombinase is provided and the transfer cassette is integrated into the target site. The polynucleotide of interest is thus integrated into a specific chromosomal position in the plant genome.

Cells that have been transformed can be grown into plants in conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can then be cultivated, and pollinated with the same transformed strain or different strains, and the resulting offspring with constitutive expression of the desired phenotypic trait identified. Two or more generations may be grown to ensure that expression of the desired phenotypic trait is stably maintained and inherited, and then seeds were harvested to ensure that expression of the desired phenotypic trait was achieved. In this way, the embodiments provide transformed seed (also referred to as "transgenic seed"), with a nucleotide construct of the embodiments, e.g., an expression cassette of the embodiments, stably incorporated into its genome.

As used herein, the term "plant" may be an entire plant, any part thereof, or a cell or tissue culture derived from a plant. Thus, the term "plant" can refer to either: the entire plant and plant components or organs (including, but not limited to, embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, seeds, ears, bark, stems, roots, root tips, anthers, and the like), plant tissues, plant cells, plant protoplasts, cell cultures of plant tissue from which the corn plant can be regenerated, plant calluses, plant clumps , seeds and plants. A plant cell is a cell from a plant, whether generated directly from a seed or plant, or derived through culture from a cell taken from a plant. Grain is mature seed produced by commercial farmers for purposes other than growth or reproduction of the species. Progeny, variants and mutants of the regenerated plants are also included within the scope of the embodiments, provided that these parts make up the introduced polynucleotides.

Embodiments of the invention can be used to confer or increase resistance to fungal plant pathogens or protect against fungal pathogen attack on plants, especially corn (Zea mays). It will protect different parts of the plant from pathogen attack, including and not limited to stems, ears, leaves, roots and tassels. Other plant species may also be of interest in practicing embodiments of the invention, including, but not limited to, other monocotyledonous plant species.

If necessary, the polynucleotides can be optimized for increased expression in the transformed organism. For example, polynucleotides can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for the synthesis of preferred plant genes. See, for example, US Patents 5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, incorporated herein by reference.

Embodiments of the present invention can be effective against a variety of plant pathogens, especially fungi, such as, for example, Colletotrichum, including Cg. Embodiments of the present invention may also be effective against corn stalk rot, including anthracnose stalk rot, in which the causative agent is Colletotrichum. Other plant pathogenic fungi and oomycetes (many of the latter have historically been considered fungi although modern taxonomists have classified them separately) include, and are not limited to, the following: Soybean: Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. soybean (Phomopsissyae), Diaporthe phaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora manshuríca,

Colletotrichum dematium (Colletotichum truncatum), Corynespora cassiicola, Septoria glycines, Phyllosticta sojicola, Alternaria alternate, Microsphaera diffusa, Fusarium semitectum, Phialophora greek ta, Glomerella glycines, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Fusarium solani; Canola: Albugo Candida, Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassiccola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Pythium ultimum, Pythium irregulare, Pythium splendens, Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronospora tri foliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium oxysporum, Verticillium alboatrum, Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfae, Colletotrichum trifolii, Leptosphaerulina briosiana, Uromyces striatus, Sclerotinia tri foliorum, Stagnospora meliloti, Stemphylium botryosum, Leptotrochila medicaginis; Wheat: Urocystis agropyri, Alternaria alternata, Cladosporium herbarum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminis f.sp. tritici, Puccinia graminis f.sp. tritici, Puccinia recondita f.sp . tritici, Pythium aphanidermatum,

Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana, Claviceps purpurea, Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletia indica, Rhizoctonia solani, Pythium arrhenomannes, Pythium gramicola, Pythium aphaniderma turn; Sunflower: Plasmophora halstedii, Sclerotinia sclerotiorum, Septoria helianthi, Phomopsis helianthi, Alternaria helianthi, Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii,

Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi, Verticillium dahliae, Erwinia carotovorum pv. carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis; Corn Fusarium moniliforme var. subglutinans,

Erwinia stewartii, Fusarium moniliforme, Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum, Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis 0, T (Cochliobolus heterostrophus), Helminthosporium carbonum I, II & III (Cochliobolus carbonum), Exserohilum turcicum I, II & III, Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis, Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi, Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvularia inaequalis, Curvularia pallescens, Trichoderma viride, Claviceps sorghi, Erwinia chrysanthemi pv. zea, Erwinia carotovora, Diplodia macrospora, Sclerophthora macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelotheca reiliana, Physopella zeae, Cephalosporium maydis, Cephalosporium acremonium; Sorghum: Exserohilum turcicum, Colletotrichum graminicola (Glomerella graminicola), Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium moniliforme, Alternaria alternate, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunata, insidious, Ramulispora sorghi , Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthona macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Sclerospora graminicola, Fusarium oxysporum, Pythium arrhenomanes, Pythium graminicola, etc. "Germplasm" refers to the genetic material of either an individual (e.g., a plant), a group of individuals (e.g., a plant strain, variety or family), or a clone derived from a strain, variety, species, or culture. Germplasm may be part of an organism or cell, or it may be separate from the organism or cell. In general, germplasm provides genetic material with a composition specific molecular basis that provides a physical basis for some or all of the heritable characteristics of an organism or cell culture. As used herein, germplasm includes cells, seeds, or tissues of young plants that can be cultivated, or parts of the plant, such as leaves , stems, pollen, or cells, which can be grown into an entire plant. The term "allele" refers to one of two or more different nucleotide sequences that occur at a specific locus.

A first allele is found on a chromosome, while a second allele occurs in the same position on a homologous chromosome, for example, as occurs across different chromosomes of a heterozygous individual, or among different homozygous or heterozygous individuals in a population. A "favorable allele" is the allele at a particular locus that confers, or contributes to, an agronomically desirable phenotype, for example, resistance to Cg infection. A favorable allele of a marker is a marker allele that segregates with the favorable phenotype. A favorable allelic form of a chromosome segment is a chromosome segment that includes a nucleotide sequence that contributes to superior agronomic performance at one or more genetic loci physically located on the chromosome segment. "Allele frequency" refers to the frequency (proportion or percentage) of an allele in a population, or of a population of lineages. One can estimate the frequency of alleles within a population by calculating the average of the allele frequencies of a sample of individuals from a specific population. 25

An allele "positively" correlates with a trait when it is linked to it and when the presence of the allele is an indicator that the desired trait or characteristic form will occur in a plant comprising the allele. An allele is negatively correlated with a trait when it is linked to it and when the presence of the allele is an indicator that a desired trait or characteristic form will not occur in a plant comprising the allele.

An individual is "homozygous" if the individual has a single type of allele at a given locus (for example, a diploid individual has one copy of the same allele at one locus for each of two homologous chromosomes). An individual is "heterozygous" if more than one type of allele is present at a given locus (e.g., a diploid individual with one copy of each of two different alleles). A special case of a heterozygous situation is where one chromosome has an allele of a gene and another chromosome is missing that gene, locus or region completely - in other words, it has a deletion of the chromosome relative to the first. This situation is referred to as "hemizygous". In this situation, the allele on the chromosome that is missing the gene, locus or region may also be referred to as the "null allele". The term "homogeneity" indicates that members of a group have the same genotype at one or more specific loci. In contrast, the term "heterogeneity" is used to indicate individuals in the group that differ in genotype at one or more specific loci.

The embodiments provide not only a gene and its functional variants for use in transgenic applications, but the sequences and processes that allow the Rcgl and Rcglb resistance genes to be moved between maize lines using marker-assisted breeding. The embodiments also relate to plants produced by these processes that maintain a truncated chromosomal interval comprising the Rcgl and Rcglb resistance genes.

A genetic map is a graphical representation of a genome (or part of a genome, such as a single chromosome), where the distances between landmarks on a chromosome are measured by the recombination frequencies between the landmarks. Recombinations between genetic landmarks can be detected by a variety of molecular genetic markers (also called molecular markers) that are described in more detail here.

For markers to be useful in detecting recombinations, they need to detect differences, or polymorphisms, within the population being monitored. For molecular markers, this means differences at the DNA level due to polynucleotide sequence differences (e.g., SSRs, RFLPs, FLPs, SNPs). Genome variability can be of any origin, for example, insertions, deletions, duplications, repetitive elements, point mutations, recombination events, or the presence and sequence of transposable elements. Molecular markers can be derived from genomic or expressed nucleic acids (e.g. ESTs). ESTs are generally well conserved within a species, while other regions of DNA (usually non-coding) tend to accumulate polymorphism and, therefore, may be more variable between individuals of the same species. A large number of maize molecular markers are known in the art, and are published or available from various sources, such as the Maize GDB internet resource and the Arizona Genomics internet resource managed by the University of Arizona.

Molecular markers can be used in a variety of plant breeding applications (e.g., see Staub et al. (1996) Hortscience 31: 729-741; Tanksley (1983) Plant Molecular Biology Reporter. 1: 3-8). One of the main areas of interest is to increase the efficiency of backcrosses and introgressed genes using marker-assisted selection (MAS). A molecular marker that demonstrates linkage to a locus that affects a desired phenotypic trait provides a useful tool for trait selection in a plant population. This is particularly true when the phenotype is difficult to assay, e.g., resistance traits to many diseases, or occurs at a late stage in plant development, e.g., kernel traits. Since DNA marker assays are less labor intensive, and take up less physical space, with field phenotyping, much larger populations can be analyzed, increasing the chances of finding a recombinant with the target segment from the donor lineage moved into the recipient lineage. The closer the link, the more useful the marker, as recombination is less likely to occur between the marker and the gene that causes the trait, which can result in false positives. Having flanking markers decreases the chances that false positive selection will occur as a double recombination event would otherwise be necessary. The ideal situation is to have a marker on the gene itself, so that recombination cannot occur between the marker and the gene. This marker is called a "perfect marker."

When a gene is introgressed by the MAS, it is not only the gene that is introduced, but also the flanking regions (Gepts. (2002). Crop Sci. 42: 1780-1790). This is referred to as "linkage drag". In the case where the donor plant is highly related to the recipient plant, as in the case of the Rcgl locus being lines generated from MP305, an exotic source, in elite lines, these flanking regions carry additional genes that may code for agronomically undesirable traits . This "linkage drag" can also result in reduced productivity or other negative agronomic traits, even after several cycles of backcrosses to the elite corn line. This is also sometimes called "yield drag". The size of the flanking region can be decreased by additional backcrosses, although this is not always successful, as breeders have no control over the size of the region or the interruption of recombination (Young et al. (1998) Genetics 120:579-585 ). In classical breeding it is generally only by chance that recombinations are selected, which contribute to a reduction in the size of the donor segment (Tanksley et al. (1989). Biotechnology 7: 257-264). Even after 20 backcrosses in backcrosses of this type, one can expect to find a considerable portion of the donor chromosome still linked to the gene to be selected. With markers, however, it is possible to select those rare individuals that have undergone recombination near the gene of interest. In 150 backcrossed plants, there is a 95% chance that at least one plant will have experienced a 1 cm backcross of the gene, based on a single meiosis map distance. Markers will allow the unambiguous identification of individuals. With an additional backcross of 300 plants, there would be a 95% chance of a cross within 1 cM of a single meiosis map on the other side of the gene, generating a segment around the target gene of less than 2 cm with based on a single meiosis map distance. This can be accomplished in two generations with markers, whereas it would have required, on average, 100 generations without markers (See Tanksley et al. supra). When the exact location of a gene is known, a series of flanking markers around the gene can be used to select for recombinations in populations of different sizes. For example, in a smaller population size recombinations might be expected further away from the gene, with more distal flanking markers being needed to detect recombination.

The availability of integrated linkage maps in the maize genome with increasing densities of public maize markers has facilitated genetic mapping of maize and MAS. See, for example, the IBM2 Neighbors 4 map [online], [visited 2006-03-21]. Taken from the Internet: <URL: http://www.maizegdb.org/cgi- bin/displaymaprecord,cgi?id=871214>.

The key components for implementing MAS are: (i) defining the population in which the trait association marker will be determined, which can be a segregating population, or a random or structured population, (ii) controlling segregation or of the association of polymorphic markers in relation to the trait, and the determination of linkage or association through statistical methods, (iii) the definition of a set of desirable markers based on the results of statistical analysis, and (iv) the use and/or extrapolation of this information to the current breeding pool of marker germplasm to allow selection based decisions to be made. The three types of markers described in this disclosure can be used in marker-assisted selection protocols; simple repeated sequence (SSR, also known as microsatellite), markers, single nucleotide polymorphism (SNP) markers, and fragment length polymorphic (FLP) markers. SSRs can be defined as relatively short runs of tandemly repeated DNA with lengths of 6 bp or less (Tautz (1989) Nucleic Acid Research 17: 6463-6471; Wang et al. (1994) Theoretical and Applied Genetics, 88:1- 6). Polymorphisms arise due to variation in the number of repeat units, probably caused by slippage during replication.

DNA (Levinson and Gutman (1987) Mol Biol Evol 4: 203-221). Variation in repeat length can be detected by designing PCR primers for conserved non-repetitive flanking regions (Weber and May (1989) Am J Hum Genet 44:388-396). SSRs are highly suitable for mapping and MAS as they are multiallelic, co-dominant, reproducible and amenable to high-throughput automation (Rafalski et al. (1996) Generation and use of DNA markers in plants. In: Non-mammalian genomic analysis: a practical guide. Academic Press, pp 75-135).

For example, an MP3 SSR marker profile 05 is provided in Example 5 here. This marker profile was generated by gel electrophoresis of amplification products generated by the primer pairs for these markers. Marker genotype score is based on the size of the amplified fragment, which in this case was measured by the base pair weight of the fragment. Although variation in the primer used or laboratory procedures may affect the base pair weight, relative values will remain constant regardless of the specific primer or laboratory used. Therefore, when comparing strains, the SSR profiles being compared must be obtained in the same laboratory, so that the same primers and equipment are used. For this reason, when comparing marker-specific plants or strains vis-à-vis, it is preferable to assert that such plants or strains have the same (or different) alleles at a given locus (e.g., one might say that if a plant does not comprise the MP305-derived chromosomal interval equal to or less than UMC15a, will not include the same alleles as MP305 at all loci at or below UMC15a listed in Table 5b in Example 5). An SSR service for corn is available to the public on a contractual basis from DNA Landmarks in Saint-Jean-sur-Richelieu, Quebec, Canada.

Various types of FLP markers can be generated. Most commonly, amplification primers are used to generate fragment length polymorphisms. Such FLP markers are, in many respects, similar to SSR markers, except that the region amplified by the primers is typically not a highly repetitive region. Still, the amplified region, or fragment, will have sufficient variability among germplasms, often due to insertions or deletions, such that fragments generated by the amplification primers can be distinguished among polymorphic individuals, and such indels are known to occur. frequently in maize (Bhattramakki et al. (2002). Plant Mol Biol 48, 539-547; Rafalski (2002b), supra). The term "indel" refers to an insertion or deletion, wherein one lineage may be referred to as having an insertion with respect to a second lineage, or the second lineage may be referred to as having a deletion with respect to the first lineage. The MZA markers disclosed herein are examples of amplified FLP markers that were selected because they are in close proximity to the Rcgl and Rcglb genes.

SNP markers detect single base pair nucleotide substitutions. Of all types of molecular markers, SNPs are the most abundant, thus having the potential to provide the highest resolution genetic map (Bhattramakki et al. 2002 Plant Molecular Biology 48:539-547). SNPs can be analyzed at an even higher level of throughput than SSRs, in a modality called "ultra high-throughput", as they do not require large amounts of DNA and automation of the assay can be taken further. SNPs also have the promise of being relatively low-cost systems. These three factors together make SNPs highly attractive for use in MAS. Various methods are available for SNP genotyping, including but not limited to, hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, mini-sequencing, and scrambled beads. These methods were reviewed in: Gut (2001) Hum Mutat 17 pp. 475-492; Shi (2001) Clin Chem 47, pp. 164-172; Kwok (2000) Pharmacogenomics 1, pp. 95-100; Bhattramakki and Rafalski (2001) Discovery and application of single nucleotide polymorphism markers in plants. In: Henry RJ, Ed., Plant Genotyping: The DNA Fingerprinting of Plants, GABI Publishing, Wallingford. A wide range of commercially available technologies utilize these and other methods to examine SNPs including MASSCODE™ (Qiagen), INVADER™ (Third Wave Technologies, Madison, WI), SNAPSHOT® (Applied Biosystems), TAQMAN® (Applied Biosystems), and BEADARRAYS™ (Lights up).

A number of SNPs together within a sequence, or across linked sequences, can be used to describe a haplotype of any specific genotype (Ching et al. (2002), BMC Genet. 3:19 pp. Gupta et al. 2001, Rafalski (2002b T), supra). Haplotypes can be more informative than single SNPs and can be more descriptive of any particular genotype. For example, a single SNP may be the T allele for MP305, but the T allele may also occur in the maize breeding population and is used for the recurrent parents. In this case, a haplotype, for example a series of marker alleles linked to the SNP, may be more informative. Once a single haplotype has been assigned to a donor chromosomal region, a haplotype can be used in the population or any subset thereof to determine whether an individual has a specific gene. See, for example, W02003054229. Using highly automated marker detection platforms known to those skilled in the art makes this process highly efficient and effective.

As described herein, many of the primers listed in Tables 1 and 2 can easily be used as FLP markers to select the Rcgl locus. These primers can also be used to convert these markers to SNP or other similar structure or functionally equivalent markers (SSRs, CAPS, indels, etc.), in the same regions. A very productive approach for SNP conversion is described by Rafalski (2002a) Current opinion in plant biology 5 (2): 94-100 and also Rafalski (2002b) Plant Science 162: 329-333. Using PCR, primers are used to amplify DNA segments from individuals (preferably innate) that represent the diversity of the population of interest. PCR products are directly sequenced in one or both directions. The resulting sequences are aligned and polymorphisms are identified. Polymorphisms are not limited to single nucleotide polymorphisms (SNP) but also include indels, CAPS, SSRs and VNTR (variable number of tandem repeats). Specifically with regard to the map information described herein, one can easily use the information contained herein to obtain more polymorphic SNPs (and other markers) in the region amplified by the primers listed in this disclosure. Markers in the described map region can be hybridized with BACs or other genomic libraries, or electronically aligned with genome sequences, to find new sequences in the same approximate location as the described markers.

In addition to SSR, FLPs and SNPs as described above, other types of molecular markers are also widely used, including but not limited to expressed sequence tags (ESTs) and SSR markers derived from EST sequences, and randomly amplified polymorphic DNA ( RAPD). As used herein, the term "Genetic Marker" refers to any type of nucleic acid-based marker, including, but not limited to, Restriction Fragment Length Polymorphism (RFLP), Simple Sequence Repeat (SSR), DNA Randomly Amplified Polymorphic (RAPD), Amplified Cleaved Polymorphic Sequences (CAPS) (Rafalski and Tingey, 1993, Trends in Genetics 9:275-280), Amplified Fragment Length Polymorphism (AFLP) (Vos et al., 1995, Nucleic Acids Res. 23:4407-4414), single nucleotide polymorphism (SNP) (Brookes, 1999, Gene 234:177-186), Characterized Sequence Amplified Region (SCAR) (Paran and Michelmore, 1993, Theor. Appl. Genet. 85:985-993), Sequence Target Site (STS) (Onozaki et al. 2004, Euphytica 138:255-262), Single Lane Confirmation Polymorphism (SSCP) (Orita et al., 1989, Proc Natl Acad Sei U.S. 86:2766-2770), Inter-Simple Sequence Repeats (fragments) (Blair et al. 1999, Theor. Appl. Genet. 98:780-792), Inter-Retrotransposon Amplified Polymorphism (TRAP), Retrotransposon Microsatellite Polymorphism -Amplified (REMAP) (Kalendar et al. 1999, Theor. Appl. Genet. 98:704-711), an RNA cleavage product (such as a Lynx tag) and the like.

More generally, the term "molecular markers" may be used to refer to a genetic marker, as defined above, or an encoded product thereof (e.g., a protein), used as a reference point in identifying a linked locus. . A marker can be obtained from genomic nucleotide sequences or from expressed nucleotide sequences (for example, from a bound RNA, a cDNA, etc.), or from an encoded polypeptide. The term also refers to nucleic acid sequences complementary to or accompanying marker sequences, such as nucleic acids used as probes or primer pairs capable of amplifying the marker sequence. A "molecular marker probe" is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus, for example, a nucleic acid probe that is complementary to a marker locus sequence. Alternatively, in some aspects, a marker probe refers to a probe of any type that is capable of distinguishing (i.e., the genotype) the particular allele that is present at a marker locus. Nucleic acids are "complementary" when they expressly hybridize in solution, for example, according to the Watson-Crick pairing rules. Some of the markers described here are also referred to as hybridization markers when located in an Indel region, such as the non-collinear region described here. This is because the insertion region is, by definition, a polymorphism vis-à-vis a plant without the insertion. Thus, the marker only needs to indicate whether the Indel region is present or absent. Any marker detection technology can be used to identify a hybridization marker, for example, SNP technology is used in the examples provided herein.

"Genomic nucleic acid" is a nucleic acid that corresponds in sequence to a hereditary nucleic acid in a cell. Common examples include nuclear genomic DNA and fragments thereof. A genomic nucleic acid is, in some cases, different from a ligated RNA, or a corresponding cDNA, in that the ligated RNA or cDNA is processed, for example, by the ligation machinery, to remove introns. Genomic nucleic acids optionally include non-transcribed (e.g., chromosomal structural sequences, promoter regions, enhancer regions, etc.) and/or untranslated sequences (e.g., introns), while bound RNA/cDNA typically have no non-transcribed sequences or introns. The "nucleic acid template" is a nucleic acid that serves as a template in an amplification reaction (e.g., a polymerase-based amplification reaction such as PCR, a ligase-mediated amplification reaction such as LCR, a transcription reaction , or something like that). A nucleic acid template may be genomic in origin, or alternatively, it may be derived from expressed sequences, for example, a cDNA or an EST.

The term "amplification" in the context of nucleic acid amplification is any process that allows additional copies of a selected nucleic acid (or a transcribed form thereof) to be produced. Typical amplification methods include various polymerase-based replication methods, including polymerase chain reaction (PCR), ligase-mediated methods such as ligase chain reaction (LCR), and RNA polymerase-based amplification methods (e.g., by transcription). An "amplicon" is an amplified nucleic acid, e.g., a nucleic acid that is produced by a method of amplifying a template nucleic acid by any available amplification method (e.g., PCR, LCR, transcription, or the like).

Isoenzyme profiles and linked morphological characteristics 5 can, in some cases, also be indirectly used as markers. Even if they do not directly detect DNA differences, they are often influenced by specific genetic differences. However, markers that detect DNA variation are much more numerous and polymorphic than isoenzymatic or morphological markers (Tanksley (1983) Plant: Molecular Biology Reporter 1:3-8).

Sequence or contig alignments can also be used to locate sequences upstream or downstream of the 15 specific markers listed here. These new sequences, close to the markers described here, are then used to discover and develop functionally equivalent markers.

For example, different physics and/or genetic maps are aligned to locate equivalent markers not described in this disclosure, but which are within similar regions. These maps can be within the corn species, or even across other species that have been genetically or physically aligned with corn, such as rice, wheat, barley and sorghum.

As noted in Example 2, by using common sequences from the region flanking the Rcgl locus that hybridized to BACs in the Mol7 and the B73 BAC libraries, BACs from both libraries were aligned with BACs from the homologous region flanking DE811ASR (BC5) of the Rcgl locus in a pathway, as shown in Figure 9(a). The public BACs B73, c0113f01, and c0117el8 were identified as directly north and south, respectively, of the Rcgl locus.

With this information, a non-contiguous extended pathway of B73 BACs between genetic markers UMC2285 and UMC15a, UMC2285 and 10 UMC2187, UMC1086 and UMC2200, or UMC2041 and UMC2200, can be created, aligning genetic markers within this region with the physical map of B73. BAC. Alignment of information on the genetic and physical maps of B73 is obtained from the Arizona Genomics Institute corn genome database on the Internet by accessing the following address with web prefix "ww.": genome.arizona.edu/fpc/maize /#webagcol. In WebChrom's view, one can select the genetic markers in the vicinity of the Rcgl and Rcglb genes and obtain a link to the physical contiguous where these genetic markers are located. By aligning the physical map of such a pathway with the genetic map one can find a multitude of B73 BACs in the region between the chromosomal intervals defined by genetic markers UMC2285 and UMC15a, UMC2285 and UMC2187, UMC1086 and UMC2200, or UMC2041 and UMC2200. BACs can be used by one skilled in the art to develop new markers for introgression of the Rcgl locus into maize germplasm. In particular, such genetic markers would be useful for monitoring the Rcgl locus in all lines in which the Rcgl or Rcgl locus and Rcglb genes have been introgressed, and for genome selection of the recurrent parent in a backcross program.

For example, to design polymorphic markers that will be useful for introgression and selection of the Rcgl and Rcglb genes, or the Rcgl locus in other maize germplasm, sequence information from the region surrounding Rcgl can be used. There are many B73 derived bacterial artificial chromosomes (BACs) available in the region of interest from which sequence information can be obtained. An example of BACs in the region of interest is shown in Figure 21, which shows a contig on the B73 physical map that is homologous to the Rcgl and Rcglb DE811ASR (BC5) region [Figure 21 retrieved 3/10/2006]. Taken from the Internet <URL: http://www.genome.arizona.edu/cgi- bin//WebAGCoL/WebFPC/WebFPC_Direct_v2.1.cgi?name=maize&contig=187 &marker=ssul>. Sequence information is obtained through information that is already publicly available (e.g., the sequence end of TAS, the sequence of Expressed Sequence Tags (ESTs) that cross the BACs in this region, the probes that are often related to these ESTs, etc.) or obtaining de novo by direct sequencing of BAC clones in this region. From this sequence you can determine which regions are most unique using various methods known to those skilled in the art. For example, using gene prediction software or by sequence comparison using BLAST® against all available maize sequence, one can select for non-repetitive sequence.

Lower copy sequencing can be used to develop a wide range of nucleic acid-based markers. These markers are used to select plant material in which the Rcgl locus is present and plant material in which the Rcgl locus is absent. If a marker outside the Rcgl locus is desired, then the markers are used to select plant material in which the Rcgl locus is present and plant material in which the Rcgl locus is absent, to determine whether the marker is polymorphic in such germplasm. Polymorphic markers are then used for marker-assisted introgression and selection of the Rcgl region and optimal selection of the recurrent parental genome, in other maize germplasm. Thus, with the location of the Rcgl locus identified and its association with Colletotrichum resistance established, one skilled in the art can utilize any number of existing markers, or readily develop new markers, which can be used to introgress or identify the presence or absence of the locus. Rcgl in the germplasm, and select for the recurrent parental genome in a backcrossing program.

In a genetic map, the linkage of a molecular marker to a gene or other molecular marker is measured as a recombination frequency. In general, the more linked two loci (e.g., two SSR markers) are on the genetic map, the closer they are on the physical map. Relative genetic distance (determined by crossover frequency, measured in centimorgans; cM) can be proportional to physical distance (measured in base pairs, e.g., kilobase pairs [kb] or mega base pairs [Mbp]) that two linked loci are separated from each other on a chromosome. The lack of proportionality between cM and physical distance may be a result of variations in the recombination frequencies of different chromosomal regions, for example, some chromosomal regions are recombination "hot spots", while other regions do not show any recombination, or only demonstrate recombination events. rare recombination. Some of the introgression and information mapping data suggest that the region around the Rcgl locus is one that has a large amount of recombination.

In general, the closer a marker is to another marker, whether measured in terms of recombination or physical distance, the more strongly they are linked. The closer a molecular marker is to the gene encoding a polypeptide that confers a particular phenotype (disease resistance), whether measured in terms of recombination or physical distance, the better marker serves to mark the desired phenotypic characteristic. If possible, the best marker is one within the gene itself, as it will always remain linked with the gene that causes the desired phenotype.

Mapping genetic variability can also be observed between different populations of the same plant species, including maize. Despite this variability in the genetic map that can occur between populations, the genetic map and marker information derived from a general population continues to be useful across multiple populations in identifying plants with desired traits, counter-selecting plants with undesirable traits. and, MAS advisor. 5

To locate equivalent markers on genetic maps, a mapping population can be used to confirm whether any equivalent markers are within the region described here and are therefore useful for selection of the Rcgl locus. Using this method, the equivalent marker, along with the markers listed here, is mapped onto the mapping of such a population. Any equivalent marker that falls within the same region can be used to select for Rcgl. Mapping populations known in the art that can be used for this purpose include, but are not limited to, the IBM populations and the T218 X GT119 IF2 population described in Sharopova, N. et al. (2002) Plant Mol Biol 48(5):463-481 and Lee, M. et al. (1999): ) : Tools for high resolution genetic mapping in maize - status report. Proc. Plant Animal Genome VII, January 17-21, 1999, 20 San Diego, USA, p. 146; the UMC 98 population, described in Davis, GL et al. (1999) Genetics 152(3):1137-72 and in Davis, MD et al., (1998) The 1998 UMC Maize Genetic Map: ESTs, Sequenced Core Markers, and Nonmaize Probes as a Foundation for Gene Discovery, Maize Genetics Conference Abstracts 40. 25

As used here, "introgression" or "introgress" refers to the passage of a gene or locus from one lineage to another: (1) the individuals interbreeding from each lineage to create a population, and (2) the selection of individuals carriers of the desired gene or locus. Selection can be done phenotypically or through markers (marker assisted selection). The individuals thus selected are again crossed (i.e., backcrossed) with the desired target lineage, and there may be two, three, four, five, six or more, or even ten backcrosses or more. After each crossing, the selection process is repeated. For example, the modality gene, or the locus containing it, may be introgressed into a recurrent parent that is not resistant or only partially resistant, which means that it is sensitive or susceptible or partially so, to Cg. The recurrent parental lineage with the introgressed gene or locus then has improved or newly conferred resistance to Cg. This lineage in which the Rcgl locus was introgressed is referred to here as a conversion of the Rcgl locus.

The process of introgression is often referred to as "backcrossing" when the process is repeated two or more times. In introgression or backcrossing, the "donor" parent refers to the parent plant with the gene or locus desired to be introgressed. "Recipient" parent (used one or more times) and recurrent parent (used two or more times) refer to the parental plant into which the gene or locus is being introgressed. For example, see Ragot, M. et al. (1995) Marker-assisted backcrossing: a practical example, in Techniques et Utilisations des Marqueurs Moleculaires (Les Collogues, vol. 72, pp. 45-56 and Openshaw et al., (1994) Marker-assisted Selection in Backcross Breeding, Analysis of Molecular Marker Data, pp. 41-43. The initial cross gives rise to the Fl generation, the term "BC1" then refers to the use of the second recurrent parent, "BC2" refers to the use of the third recurrent parent, and so on. against.

In the case of the Rcgl locus, where gene sequences responsible for its phenotypic effect and closely related regions are available, DNA markers based on its genes or closely linked sequences can be developed for direct selection of donor genes in the recurrent parent. Although any polymorphic DNA sequence from the chromosomal region carrying the gene can be used, the predicted sequences in the embodiments allow the use of DNA markers within or near the genes, minimizing false positive selection for the genes. Flanking markers limit the size of the donor genome fragments introduced into the recipient, thus minimizing so-called "linkage drag", which means the introduction of undesirable sequences into the donor lineage that could impact the plant's performance against elite germplasm. The developments provide several examples of DNA markers that could be used, and one skilled in the art will be able to use the gene sequences provided to create even more markers. An example is the use of markers that cross (in the case of RFLP assays) or ring (in the case of PCR), specifically (exclusively) closely linked sequences, including within the locus. In principle, sequences that also cross or anneal elsewhere in the genome could be used if several markers are used in combination. When PCR reactions are used, in practice the length of the primers used in the amplification reaction should be at least about 15 nucleotides, but depending on the sequences and hybridization conditions, any length that provides all the specific annealing can be used. , like about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28 or so. For PCR reactions the term "annealing" is commonly used, and as herein it should be used and understood to have the same meaning as "crossover".

Thus, using the markers and processes described herein, one can produce a plant comprising a truncated chromosomal interval comprising the Rcgl locus and/or the Rcgl and Rcglb genes. The term "chromosomal gap" or "chromosomal segment" refers to a short linear contiguous distance of genomic DNA that resides in the plant on a single chromosome, generally defined by reference to two markers defining the endpoints of the chromosomal gap. The specified range may include markers at endpoints (e.g., one or more markers within the chromosomal interval defined by marker A and marker B) or may exclude markers at the endpoints of a range (e.g., one or more markers within the chromosomal interval defined by marker A and marker B). A truncated chromosomal interval refers to a chromosomal interval that has been reduced in size by selecting for one or more recombination events that reduced the size of the chromosomal interval. A "recombination event" refers to the occurrence of recombination between homologous chromosomes, and refers to a specific chromosomal location where such recombination has occurred (e.g., a recombination of an internal chromosomal interval to chromosome end points will have a recombination event at each end of the chromosomal interval). The truncated chromosomal interval can be defined by reference to one or two new markers at the end points of the segment. The length of the two chromosome segments can be measured by either centimorgans or base pairs. The genetic elements or genes located in a single chromosomal interval are physically linked. The size of a chromosomal interval is not particularly limited, but in the context of embodiments of the present invention, generally genetic elements located in a single chromosomal interval are also genetically linked.

By using the methods of the embodiments, it is possible to select for a plant comprising a truncated chromosomal interval comprising the Rcgl and Rcglb genes. Specifically, with respect to the invention described in more detail in the examples below, the chromosomal interval may be reduced to a length of 12cM or less, 10cM or less, 8cM or less, 6cM or less, 4cM or less, 3 cM or less, 2.5 cM or less, 2 cM or less, 1.5 cM or less, 1 cM or less, 0.75 cM or less, in each case measured relative to map distances as shown in the IBM2 Neighbors 4 genetic map effective March 21, 2006. As measured in base pairs, the chromosomal interval can be reduced to a length of 15 mbp or less, 10 mbp or less, 5 mbp or less, 3 mbp or less, 1 mpb or less, 500 kbp or less. One skilled in the art would understand that it is undesirable to cause a disruption in the chromosomal region so proximal to the Rcgl or Rcglb coding sequences (e.g., within 5 kbp or less, within 4 kbp or less, 3 kbp or less, 2 kbp or less, less, 1 kbp or less), such that the promoter and other regulatory elements above would be unlinked from the coding sequence.

The term "locus" generally refers to a genetically defined region of a chromosome carrying a gene or, possibly, two or more genes so closely linked that genetically they behave as a single locus responsible for a phenotype. When used here with respect to Rcgl, the "Rcgl locus" refers to the defined region of the chromosome carrying the Rcgl and Rcglb genes including their associated regulatory sequences, plus the region around the two genes that is not colinear with B73, or any minor part thereof, that carries the Rcgl and Rcglb genes and associated regulatory sequences. This locus has also been referred to elsewhere as the ASR locus, and will be referred to here as the Rcgl locus.

A "gene" refers to a specific genetic coding region within a locus, including its associated regulatory sequences. The coding region of the Rcgl primary transcript, referred to herein as the "Rcgl coding sequence", will be used to define the position of the Rcgl gene, and one skilled in the art would understand that the associated regulatory sequences will be within a distance of about 4 kb from the Rcgl coding sequence, with the promoter located upstream. The coding region of the Rcglb primary transcript, referred to herein as the "Rcglb coding sequence", will be used to define the position of the Rcglb gene, and one skilled in the art would understand that the associated regulatory sequences will be within a distance of about of 4 kb (i.e., about 4 kb, about 3 kb, about 2 kb, about 1 kb, or about 0.5 kb) of the Rcglb coding sequence, with the promoter located upstream. of the present invention is the isolation of the Rcgl and Rcglb genes and the demonstration that they together are the genes responsible for the phenotype conferred by the presence of the locus.

As will be appreciated by one skilled in the art, the structure of the Rcgl locus brings with it special considerations when using MAS to work with this locus. It comprises two genes (Rcgl and Rcglb), located within less than a centimorgan of each other that are each necessary for the phenotype conferred by the presence of the locus. Physically the two genes are about 200 - 300 kb apart. This means that the chances of recombination between the two genes, so that they are separated, is very low, which means less than 1%, on a chromosome the size of chromosome 4 (about 250 megabases, or 250,000 kb). There are several reasons for this.

1. The physical distance represents about 0.1% (i.e., as a fraction 0.001) of the chromosome length. If recombination occurs randomly, the probability of a recombination event in this region is clearly low.

2. While recombination frequencies are not identical at different locations in the genome, the average physical distance per centimorgan (1% recombination - see Suzuki, Griffiths, Miller, and Lewontin, (1986) An Introduction to Genetic Analysis, Third Edition, Freeman & Company, New York, page 84) is about 1,000 kb. 200-300 kb thus represents less than 1 cM, or less than 1% recombination. The number of recombination events found in Example 1 in the general region flanking the locus is not particularly high.

3. As shown in Example 2, the locus is present in a chromosomal region of MP305 that is not present in DE811 and indeed in many other strains used as breeding sources for commercial strains. This region is referred to here as the non-collinear region. In these cases, there may be no recombination because there is no homologous region on the other chromosome. Consistent with this, in Example 1, recombination events were found only outside the region introgressed from MP305. This specific reasoning applies only to those strains that do not contain a segment homologous to the MP305 region, but one skilled in the art will easily be able to determine this using the methods described in Example 11.

Taken together, this means that although, in principle, to avoid any possibility of separating the two genes, one should use markers outside the range defined by the two genes, as a practical matter one could use markers within either gene, or in the interval between them, to track and successfully introgress both genes. The statistical possibility of missing one or the other gene is less than 1%, as described above, and even the risk can be avoided with various analyzes to detect the presence of individual genes (including but not limited to gene-specific PCRs, RT- PCR, westerns, ELISA and other methods mentioned above). Therefore, embodiments of the present invention include both methods for using MAS in a way that avoids any risk of missing one or the other gene, using markers flanking the range defined by the genes, but also methods based on following just one or the other gene. , or the region between them, combined with the appropriate dosages to make sure both genes are present.

It is also important to note that, because the two genes are so closely linked, previous researchers would not have observed them as two genes. Thus, these two genes do not represent the two genetically separable genes on the long arm of chromosome 4 that confer resistance to Cg reported previously (Toman, et al., (1993), Phytopathology, 83:981-986; Cowen, N et al. (1991) Maize Genetics Conference Abstracts 33). The two genes together at the Rcgl locus of the present invention may represent one of two genetically separable loci, which have been previously reported, but which have not yet been associated with a particular gene or finely mapped locus.

As used herein, "linked" or "linkage" (as distinguished from the term "operationally linked") refers to the genetic or physical linkage of loci or genes. Loci or genes are considered genetically linked if the frequency of recombination between them is less than about 50%, as determined in a single meiosis map. They are increasingly linked, whether the recombination frequency is about 40%, about 30%, about 20%, about 10% or less, as determined in a single meiosis map. Two or more genes are physically linked (or syntenic) if they have been shown to be on a single piece of DNA, such as a chromosome. Genetically linked genes, in practice, are physically linked (or syntenic), but the exact physical distance (number of nucleotides) may not have been demonstrated yet. As used herein, the term "closely linked" refers to markers genetically linked within 15 cM or less, including, without limitation, 12 cM or less, 10 cM or less, 8 cM or less, 7 cM or less, 6 cM or less, 5 cM or less, 4 cM or less, 3 cM or less, 2 cM or less, 1 cM or less, and 5 cM or less, as determined from the publicly available IBM2 neighbors 4 genetic map on the Maize GDB website previously mentioned in this disclosure. Distances of 1 cM or less and 0.5 cM or less can also be considered very close, or strongly linked. DNA sequence, such as a short oligonucleotide representing a sequence within a locus or one complementary to it, is also linked to that locus.

A "lineage" or "strain" is a group of individuals of identical ancestry who are generally pure to some extent and who are generally homozygous and homogeneous at most loci.

An "ancestral line" or "progenitor line" is a parental line used as a source of genes, for example, for the development of elite lines. "Progenies" are the descendants of the ancestral line, and may be separated from their ancestors by several generations of breeding. For example, many elite lines are the descendants of B73 or Mol7. A "pedigree structure" defines the relationship between a descendant and each ancestor that gave rise to that descendant. The pedigree structure may span one or more generations, describing the relationships between descendants and their parents, grandparents, great-grandparents, great-great-grandparents, etc.

An "elite line" or "elite variety" is an agronomically superior line or variety that has resulted from many cycles of breeding and selection for superior agronomic performance. A "pure elite line" is an elite line that is pure, and that has been shown to be useful for producing sufficiently high-yielding and agronomically high hybrid varieties (a "hybrid elite variety"). Numerous elite lines and varieties are available and known to those skilled in the art of corn production. Likewise, "elite germplasm" is an agronomically superior germplasm, typically derived from and/or capable of giving rise to a plant with superior agronomic performance, such as an existing or newly developed elite line of maize.

In contrast, an "exotic corn line" or "exotic corn germplasm" is germplasm derived from corn that does not belong to an available elite line, elite variety of germplasm, or elite germplasm. In the context of a cross between two corn plants, an exotic line or exotic germplasm is not closely related to the offspring until the elite line, elite variety or elite germplasm with which it is crossed. Most commonly, the exotic lineage or exotic germplasm is marked to introduce new genetic elements (typically new alleles) into a breeding program.

Units, prefixes and symbols can be noted in their accepted SI forms. Unless otherwise noted, nucleic acids are written left to right in a 5' to 3' orientation; Amino acid sequences are written from left to right in an amino acid to carboxyl orientation, respectively. Numeric ranges are inclusive of the numbers that define the range. Amino acids may be referred to here by one or another of the three known symbols or by letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes. The terms defined above are more fully defined by reference to the specification as a whole.

Regarding map directions here, instead of terms 5' and 3', the terms "north" and "above" are used (e.g. a marker north of the Rcgl locus refers to a marker above the Rcgl locus, as determined with reference to maps provided in a vertical orientation, as in Figures 7 and 8, and to the left of the Rcgl locus, determined with reference to maps provided in a horizontal orientation, as in Figure 22). Likewise, the terms "South" and "below" are used (e.g., a marker south of the Rcgl locus refers to a marker below the Rcgl locus, determined with reference to the vertical orientation maps envisioned here, and to the right of the Rcgl locus, determined with reference to the horizontal orientation maps provided here). More specifically, for example, above the Rcgl coding sequence refers to the chromosome above, or north of the primary transcript in SEQ ID NO: 1 (at about FLP110F), and below the Rcgl coding sequence refers to the chromosome below or south of the primary transcript in SEQ ID NO: 1 (at about FLPA1R). See Figure 26. The terms "proximal" and "distal" are terms relating, respectively, to the closest and most distant to a given location, for example, (the Rcglb or Rcgl genes), when used to compare two points on a map relative to the specified location.

A phenotypic assay is an assay in which, rather than using molecular markers to follow a trait or characteristic, the plant's phenotype with respect to the trait or characteristic is verified. For any data or characteristic trait, the technician will be able to develop or find numerous phenotypic assays in the literature. For example, two phenotypic resistance assays for Colletotrichum graminicola are the leaf burn tests described in Example 12, the stem infection assay described in Example 14. Other phenotypic resistance assays for Colletotrichum assays are possible. The term "computer systems" generally refers to various automated systems used to perform some or all of the steps of the method described herein. The term "instructions" refers to computer code that instructs the computer system to perform some or all of the steps of the method.

In addition to practicing some or all of the steps of the method, digital or analog systems, for example, include a digital or analog computer, can also control a variety of other functions, such as visible display to the user (e.g., to allow viewing of method results by a user) and/or control of resource output (e.g. to assist in assisted selection or control of automated field equipment).

Some of the methods described in this document are optionally (and typically) implemented through a computer program or programs (e.g., that store and can be used to analyze molecular marker data). Thus, the embodiment provides digital systems, e.g., computers, computer support means and/or integrated systems that include instructions (e.g., integrated into suitable programs) for carrying out the methods thereof. The digital system will include information (data) corresponding to plant genotypes for a set of genetic markers, and, optionally, phenotypic values and/or family relationships. The system can also assist the user in performing assisted selection for the Rcgl locus according to the methods herein, or can control field equipment that automates selection, harvesting and/or breeding systems.

Standard desktop applications such as word processing software (e.g., Microsoft ® Word or Corel ® WordPerfect ®) and/or database software (e.g., spreadsheet software such as Microsoft ® Excel ®, Corel ® Quattro Pro ®, or database such as Microsoft ® Access ™ or Paradox ®) can be adapted for the materialization of input data that is loaded into the memory of a digital system, and perform an operation as observed here in the data. For example, systems may include upstream software with appropriate genotype data, and optionally, pedigree data, used in conjunction with a user interface (e.g., a GUI on a standard operating system, such as a Windows®, Macintosh ® or Linux ®) to perform any analysis mentioned here, or simply to acquire data (for example, in a spreadsheet) to be used in the methods here. The computer may be, for example, a PC (Intel® x86 or Pentium® chip compatible with MS-DOS®, OS/2®, Windows®, Windows NT®, Windows 95®, Windows® 98, Linux®, compatible with an Apple®, Macintosh® compatible, POWER PC® compatible or a UNIX® compatible (e.g., SUN™ workstation) machine) or other commercially common computer that is known to one skilled in the art. Software for association analysis and/or phenotypic value prediction can be constructed by one skilled in the art using a standard programming language such as Visual Basic®, Fortran®, Basic, Java®, or similar, in accordance with the methods herein .

Any system controller or computer optionally includes a display, which may include, for example, a cathode ray tube (CRT), a flat panel display (e.g., active liquid crystal array display, liquid crystal display) , or others. Computer circuitry is often placed in a box that includes several integrated circuit chips, such as a microprocessor, memory, interface circuits, and so on. The box also optionally includes a hard disk drive, a floppy disk drive, a high-capacity removable drive such as a writable CD-ROM, and other common peripheral elements. Introducing devices such as a keyboard or mouse optionally provide a user input and user selection of the genetic marker genotype, phenotypic value, or the like into the relevant computer system. The computer generally includes software suitable for receiving instructions for use, either in the form of user input into a set of parameter fields, e.g. in a GUI, or in the form of pre-programmed instructions, e.g. -Programmed for a variety of different specific operations. The software converts these instructions into an appropriate language to instruct the system to perform any desired operation. For example, a digital system can instruct the selection of plants comprising certain markers, or field control machines for harvesting, selecting, crossing or preserving crops according to the relevant method.

The invention may also be incorporated within an application-specific integrated circuit (ASIC) or programmable logic device (PLD). In this case, the invention is embodied in a computer descriptor language that can be used to create an ASIC or PLD. The invention may also be incorporated within the logic circuit or transformers of a variety of other digital devices, such as PDAs, portable computer systems, monitors, image editing equipment, etc.

Examples

Embodiments of the invention are also defined in the following examples, where all parts and percentages are by weight and degrees are Celsius unless otherwise indicated. It should be understood that these examples, although indicating embodiments of the invention, are given by way of illustration. From the above discussion and these examples, one skilled in the art can know the essential characteristics of the embodiments of the present invention, and without departing from the scope and claims, can make various changes and modifications to adapt it to different uses and conditions. Thus, various modifications of embodiments of the invention in addition to those shown and described will be apparent to those skilled in the art from the above description. Such changes are also intended to fall within the scope of the attached claims. The disclosure of each reference contained herein is incorporated by reference in its entirety. Examples 5 and 15 are partly real and partly prophetic. All other examples are real.

Example 1

Fine mapping of the Rcgl locus to a specific region of 4L In order to map and clone the gene responsible for the resistance of the maize line to MP305 to Cg, lines had previously been created which differed as little as possible from each other genetically with the exception of presence of the locus responsible for the resistant phenotype. Such strains are called isogenic strains. To this end, DE811 had been crossed with MP305 and the progeny had been backcrossed to the sensitive line DE811 three times, in each backcross selecting for resistance to Cg and another for the characteristics of DE811 (Weldekidan and Falcão (1993), Maydica, 38 :189-192). The resulting strain was designated DE811ASR (BC3) (Weldekidan and Hawk (1993 supra)). This lineage was used as a starting point for fine mapping of the Rcgl locus. It was first necessary to know approximately where in the maize genome it was located. Using standard genetic methods, Jung et al. ((1994 supra)) had already located the locus on the long arm of chromosome 4.

Since the Rcgl locus had already been mapped on the long arm of maize chromosome 4, using information about markers near the location obtained by Jung et al. (1994) supra, all available public and private simple sequence repeats (SSR) markers located in a region of the chromosome designated 4.06-4.08 were analyzed to determine whether these markers were polymorphic between the two isogenic lineages close to DE811 and DE811ASR (BC5). DE811ASR (BC5) was derived from DE811ASR (BC3), a strain described by Weldekidan and Hawk (1993), supra through two backcrosses to DE811 under selection for Cg resistance, followed by 5 generations of selfing and selection to obtain the BC5 strain. . The BC5 line was backcrossed two more times to DE811 to create BC7 segregating the population used for fine mapping. In order to be able to conduct phenotypic assessment on a family basis, BC7 individuals were self-fertilized to create BC7S1 families.

From this analysis two SSR markers, PHI093 and UMC2041, were discovered to be polymorphic. Using the intermated (Coe et al. (2002) Plant Physiol. 128:9-12; Gardiner, et al., (2004) Plant Physiol., 134:1317-1326; Yim et al., (2002) Plant Physiol. 130:1686-1696) B73 , the three restriction fragment length polymorphism (RFLP) sequence tags, CDO365, CSU166, and CDO127, were used to create fragment length polymorphic tags (hereinafter referred to as FLPs). FLPs are markers that can be analyzed by gel electrophoresis or similar high-resolution fragment separation method followed by a PCR reaction using primers of a defined sequence. All three markers were found to be polymorphic. The FLPs used in mapping the Rcgl locus are summarized in Table 1. Any primer for the MZA FLPs presented in Table 1, which also have the same MZA marker names presented in Table 2, will amplify a region of the FLP internal to the internal sequence in Table 2. The annealing temperature for all initiators listed in Table 1 is 60°C.

In order to determine whether the presence of these three polymorphic FLPs and two polymorphic SSRs was associated with the resistance phenotype, indicating that the region carrying the Rcgl locus was located on a chromosomal segment containing these three markers, a table was created in which the status Phenotypic status of 4784 individuals determined by field observation and the genotypic status relative to each of the five markers, determined by fragment size analysis, were enrolled. These data were presented sequentially to the software programs JoinMap (Van Ooijen, et al., (2001), Plant Research International, Wageningen, the Netherlands) and Windows QTL Cartographer (Wang, et al., (2004), (on- line, version 2.0 retrieved on 6/14/2004 and version 2.5 visited on 2/22/2005); retrieved from the North Carolina State University Statistical Genetics and Bioinformatics website <URL: http://statgen.ncsu.edu/qtlcart /WQTLCart.htm>. The former program determines the order of markers along the chromosomal region. The latter determines whether a particular allele of a marker (a special form of two polymorphic forms of the marker) is significantly associated with the presence of the phenotype. Markers for which the presence of one or the other allele is more significantly associated with the resistance phenotype are more likely to be closer to the gene responsible for the resistant phenotype. Figure 3 shows a graph produced by Windows QTL Cartographer showing a statistical analysis of the resistance phenotype. chance (Y axis) where the locus responsible for the Cg resistance phenotype is located at a certain position along the chromosome (X axis), as defined by the FLP markers.

From the integrated physical and genetic map, as described by Fengler, et al., ((2004) Plant and Animal Genome XII Abstract Book, Page 192 (Poster number P487), January 10-14, San Diego, California) and supra Gardiner, (2004), it was possible to identify two contiguous bacterial artificial chromosomes (BAC), derived from a Mol7 BAC library, harboring the above-mentioned genetic markers.

However, the two contiguous BAC containing the markers flanking the region of interest contained a difference of unknown size. In order to identify more BACs to bridge this gap, a dense genetic map containing markers (Fengler, (2004 supra)) with known positions on the physical map was used to find additional markers genetically linked to the previously identified markers in the two contiguous BACs. These additional markers in Table 2 were used to identify contiguous BACs from a BAC B73 library, which decreased the physical difference between the previously found, contiguous Mol7 derived BACs (Coe et al. (2002) supra; Gardiner (2004) supra; Yim et al. (2002) supra. Four markers, MZA11455, MZA6064, MZA2591 and MZA15842, were used for mapping purposes, respectively. , to the outer and inner primers used during the nested PCR. The outer set is used in the first round of PCR, after which the inner sequences are used for a second round of PCR on the products from the first round. Capital letters indicate primer parts based on vector sequences, which are subsequently used to sequence the PCR product. ID NO: 126, and for reverse internal nested MZA primers, the top is SEQ ID NO: 127. The sequences presented in Table 2 for the internal nested MZA primers are therefore a combination of SEQ ID NO: 12 6 plus a SEQ ID NO: for each respective primer. Likewise, the sequences presented in Table 2 for reversing the nested MZA internal primers are a combination of SEQ ID NO: 127 plus SEQ ID NO: for each respective primer. These combinations are indicated in the SEQ ID NO: column of Table 2. The annealing temperature for all primers listed in Table 2 is 55° C. All markers set forth in Table 2 showed polymorphism within a diverse panel of maize germplasm, including MP305 and the maize lines in Table 18.

Sequences from the ends of several of these BACs, as well as ESTs known to be located on these BACs, were used to identify new markers with which to further narrow the range in which the locus was located. The most commonly used markers for this purpose are designated FLP8, FLP27, FLP33, FLP41, FLP56 and FLP95 in Table 1. In a similar way to those described above, genotypic and phenotypic correlations were made. It was determined that the locus was most likely located between FLP 8 and FLP 27 (see Figure 3).

Example 2

Isolation of BAC clones from resistant strains and identification of candidate genes in the Rcgl locus region

In order to isolate the gene responsible for the phenotype conferred by the Rcgl locus, BACs containing the region between markers FLP 8 and FLP 27 were isolated from a BAC library prepared from the resistant strain DE811ASR (BC5). This library was prepared with standard techniques for the preparation of genomic DNA (Zhang et al. (1995) Plant: Journal 7:175-184) followed by partial digestion with HindIII and size ligation of selected fragments into a commercially available modified form of the vector pCClBAC™ (Epicentre, Madison, USA). After transformation into EPI300™ E. coli cells following the sellers' instructions (Epicentre, Madison, U.S.A.), 125,184 recombinant clones were arrayed on 326 384-well microtitered discs. These clones were then gridded on nylon filters (Hybond ® N +, Amersham Biosciences, Piscataway, USA).

The library was probed with overlapping oligonucleotide probes (overgo probes; Ross et al. (1999) Screening large-insert libraries by hybridization, p. 5.6.1-5.6.52, Tn A. Boyl, ed. Current Protocols in Human Genetics. Wiley, New York) designed from sequences found in the BAC sequences shown in the previous example to be present between FLP8 and FLP27. BLAST™ search analyzes were performed to display repeated sequences and identify sequences unique to probe design. The position and spacing of the probes along the contig were verified by PCR. For each test two complementary 24-mer oligos longer than 8 bp were designed. Its annexes resulted in an overgo of 40bp, of which two 16bp were filled. The probes used in this way are shown in Table 4. Note that some of these tests were based on markers also used in Example 1 and Table 1, but the exact sequences are different as they were being used as probes rather than just PCR primers. . Probes for hybridization were prepared as described (Ross et al. (1999 supra)), and filters prepared by screening the BAC library were hybridized and washed as described by (Ross et al. (1999 supra)). Phosphorimaging analysis was used to detect hybridization signals.

Subsequently, the membranes were stripped of probes by placing them in a single solution of 0.lx SSC and 0.1% SDS and allowing them to cool to room temperature in the solution overnight. BACs that gave a positive signal were isolated from the plates. Restriction mapping, PCR experiments with primers corresponding to the previously used markers, and sequences obtained from the ends of each BAC were used to determine the order of BACs covering the region of interest. Four BACs that covered the entire region were selected for sequencing. These BACs were sequenced using standard bombardment sequencing techniques and assembled sequences using the Phred/Phrap/Consed software package (Ewing et al. (1998) Genome Research, 8:175-185).

After assembly, sequences known to be in the region proximal to the locus from the mapping data were annotated, meaning that possible gene-coding regions and those representing repetitive elements were inferred. Gene-coding regions (genes) were evaluated using the FGENESH™ software package (Softberry, Mount Kisco, NY, USA). FGENESH™ predicted a portion of a protein that, when compared using BLAST® (BLASTx/nr), exhibited partial homology at the amino acid level to a portion of a rice protein that was annotated as encoding a protein that confers disease resistance in rice. The portion of the maize sequence that exhibited homology to this protein fell at the end of a contiguous stretch of the BAC consensus sequence and appeared to be truncated. In order to obtain a complete representation of the maize BAC gene, the rice amino acid sequence was used in a tBLASTn analysis against all other consensus sequences from the same maize BAC clone. This resulted in the identification of a consensus sequence representing the 3' end of the maize gene. However, the central portion of the gene was not represented in the sequences thus obtained. PCR primers were designed based on the 5' and 3' regions of the putative gene and used in a PCR experiment with DNA from the original maize BAC as a template. The sequence of the PCR product was constructed by bridging the 5' and 3' sequence of previously isolated fragments. DE811ASR (BC5) has been deposited with the ATCC, and the methods described in this document can be used to obtain a BAC clone comprising the Rcgl locus. As shown in Figure 9(a), the chromosomal interval DE811ASR (BC5) with the Rcgl locus is non-collinear with the corresponding region of B73 and Mol7 (see Figures 9 and 22), as determined by analysis of BAC libraries.

Using common sequence that crosses the BACs in the Mol7 and the B73 BAC libraries, the corresponding BACs from both libraries were queued in a path, as shown in Figure 22. The B73 BACs in Figure 22 were given shorter names for the purposes of the figure. . Table 3, below, shows the BAC identity for each BAC designation indicated in Figure 22. The public BACs B73, c0113f01, and c0117el8 are directly north and south, respectively, of the Indel region of the Rcgl locus, with suppression occurring in B73. Information about these two BACs can be seen on several websites such as the GDB corn website (maizegdb.org), the Gramene website (gramene.org), and the Arizona Genomics Institute corn genome database (genome.arizona.edu). . The Arizona Genomics Institute website also provides the Corn Agarose FPC Map, version dated July 19, 2005, which identifies BACs contiguous with c0113f01 and c0117el8. By searching these databases, a multitude of contiguous BACs were identified that form one of the regions flanking the Rcgl locus. Thus, the precise location of the Rcgl locus and Rcgl gene have now been identified on both the genetic and physical map of maize. See Figures 7(a,b) and 22. Table 3: BAC designations in Figure 22 that were part of either contig 187 (B73a through B73p) or contig 188 (B73q through B73af) of B73as shown on the Arizona Genomics website Institute mentioned above.

The complete sequence of the putative gene is set forth in SEQ ID NO: 1. The gene contains an intron, from nucleotide 950 to nucleotide 1452 of SEQ ID NO: 1. Reverse transcriptase-PCR 5 using RNA prepared from DE811ASR (BC5) plants was used to determine intron boundaries. The gene-encoding protein sequence is set forth in SEQ ID NO: 2, and the amino acid translation is set forth in SEQ ID NO 3. The predicted protein has a molecular weight of 110.76 kD.

The amino terminus of about 157 to 404 amino acids has homology to the so-called nucleotide binding sites (NBS). There is a loose region of homology to LRR domains located approximately from amino acids 528 to 846. However, unlike previously studied NBS-LRR proteins, the leucine-rich region lacks the systematic repetitive nature (Lxx) found in more classical LRR domains and, in particular, lacks the instances of consensus sequences described by Wang et al. ((1999), Plant J. 19:55–64) or Bryan et al. ((2000), Plant 10 Cell 12:2033–2045). The gene has lost homology to a family of rice genes and a barley gene, as shown in Figure 2(a, b and c). Most of the homology is at the amino terminus of the protein, the carboxyl terminus being quite different. This is demonstrated by the use of bold in Figure 2 15(a, b and c) , which are the amino acids identical to the gene embodiments, while those that are not identical are not shown in bold. Table 4. Annealed oligonucleotides to synthesize the probes

Example 3

Comparison of the genetic structure of the Rcgl locus region between resistant and susceptible lines and expression profiles of candidate genes found in the region between resistant and susceptible lines

Having found a candidate gene from the genetically defined region to carry the locus responsible for the anthracnose resistance phenotype, efforts were undertaken first to determine whether there might be other genes present in the region and second, to determine whether the expression patterns of the candidate gene were consistent with its putative role. Fu and Dooner ((2002), Proc Natl Acad Sci 99:9573-9578) and Brunner et al. ((2005), Plant Cell 17:343-360) have shown that different maize inbred lines can have significant rearrangements and lack of collinearity with respect to each other. Comparison of genomes, such as those in larger regions, can thus be complex. This comparison of the genomes of Mol7 (Missouri 17) and DE811ASR (BC5) revealed that in the region where the candidate gene is found in DE811ASR (BC5), a large insertion relative to Mol7 is present. Regions within and around the insertion were sequenced and scanned for possible genes. A gene encoding a subunit of ribulose bisphosphate carboxylase (Rubisco, a protein involved in carbon fixation after photosynthesis, the gene for which is present in multiple copies in the maize genome) was found in both the DE811ASR(BC5) and Mol7 genomes, just downstream of the Rcgl gene position. A pseudogene (a gene rendered nonfunctional due to coding sequence mutations) related to a vegetative storage protein was found, present only in the DE811ASR(BC5) genome, some distance upstream of the Rcgl gene. The only structurally intact gene likely to encode a protein with a probability function of being related to disease resistance was the Rcgl gene isolated in the previous example. Other genes equally unlikely to be involved in disease resistance were located at a greater distance from the most likely position of the locus, as were a large number of repetitive sequences.

In order to determine if and where the Rcgl gene was transcribed, two techniques were used. First, the RNA profiles of resistant and susceptible plant materials were examined using Link-and-Slice-Based Parallel Signature Sequencing (MPSS™; Lynx Therapeutics, Berkeley, U.S.A.). Briefly, cDNA libraries were constructed and microimmobilized as described (Brenner, S. et al. (2000) Nat. Biotechnol. 18 (6): 630-634). Construction of the library on a solid support allows the library to be arrayed in a monolayer and thousands of clones to be subjected to nucleotide sequence analysis in parallel. The analysis results in a 17-mer sequence "signature" whose frequency of occurrence is proportional to the abundance of that transcript in plant tissue. cDNA derived from RNA prepared from DE811ASR (BC5) and DE811 (control lines, susceptible to Cg) was subjected to analysis by MPSS™. Bioinformatics inspection of the resulting signatures showed that a signature sequence, referred to herein as Lynxl9, (SEQ ID NO: 19) was present at 43 parts per million (ppm) in DE811ASR (BC5) RNA samples from infected stems and at 65 ppm in infected, from resistant stems 9 days post-inoculation (DPI) with Cg. This signature sequence was not detected in cDNA libraries from infected or Cg- infected stems of DE811 maize from susceptible lines. An analysis of the Rcgl sequence indicates that mer-17 is present at nucleotides 3945-3961 of SEQ ID NO:. 1 in the 3' untranslated region of the putative gene.

Further evidence that Rcgl is exclusively expressed in maize lines that are derived from MP305 and resistant to anthracnose stalk rot was obtained by RT-PCR. Total RNA was isolated from infected and Cg- infected stems of resistant (DE811ASR1 (BC5)) and susceptible (DE811) infected lines of maize using RNA STAT™"60 (Iso-Tex Diagnostics, Dallas, Texas, U.S.A.). Total RNA (250 ng) of 0, 3, 6, 9 and 13 DPI from resistant and susceptible samples was copied into DNA and amplified using a GeneAmp ® RNA-PCR kit (Applied Biosystems, Foster City, California, USA). DNA synthesis was assembled according to the protocol kit using random hexamers as primers and incubated at 42 2C for 45 minutes. For PCR, KEB131 (SEQ ID NO: 20) and KEB138 (SEQ ID NO: 21), both designed. from the 3' untranslated sequence of the putative Rcgl, were used as upstream and downstream primers, respectively. DNA was amplified for 30 cycles consisting of 1 minute at 94°C, 2 minutes at 50°C, and 3 minutes. at 72° C, followed by a 7-minute extension at 72° C. As shown in Figure 4, gel electrophoresis of an aliquot of the RT-PCRs revealed the presence of a 260 bp band present in samples from both plants resistant infected and uninfected, but absent from sensitive samples. DNA sequence analysis confirmed that this fragment corresponded to nt 3625-3884 of the Rcgl sequence consistent with the predicted amplification product from primers KEB131 and KEB138.

Example 4

Isolation of strains containing Mu insertions in the candidate gene

One method of determining whether a gene is responsible for a phenotype is to disrupt the gene genetically by inserting a transposable element (so-called transposon tagging) and then determine whether the relevant plant phenotype is altered, in this case from Cg resistance to Cg susceptibility. In maize this can be done using the modifier (Mu) element (Walbot, V. (1992) Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:49-82). The basic strategy shown in Figure 5 was to introduce active mutator elements into lines carrying the resistance gene, isolate plants homozygous for the resistance gene by analyzing associated DNA markers as well as Cg resistance by inoculation with Cg, and then cross these homozygous plants with a susceptible "check" line. If the resistance gene is dominant, in principle all resulting progeny would be resistant but heterozygous for the gene. However, if a Mu element inserted into the resistance gene in a way that disrupted its function, that individual would be susceptible to Cg. The disrupted gene could then be isolated and characterized.

MP305 was crossed with fifteen diverse mutator units (lines carrying active mutator elements). The resulting Fls were inter-mated (crossed with each other) in every possible combination. To track the 4L chromosomal region in which the resistance locus was known (see Example 1) a variety of DNA markers to be known in the vicinity of the locus from the work described in Example 1 were selected and used in the Mu-labeled materials. 1500 plants descended from the inter-mating process were examined for Cg resistance and the presence of these markers. Marker analysis was done using Southern blots (Botstein et al., (1980) Am. J. Hum. Gen. 32:314-331) for RFLP markers or PCR for FLP markers, as described in example 1. Plants that were homozygous for all markers tested and resistant to Cg were selected and backcrossed with susceptible test lines (A63, EH6WA and EF09B). About 16,000 cross seeds generated from these homozygous, resistant plants were planted and were used as female parents (meaning pollen production with edges removed) and crossed with the sensitive test lines used as males. All female plants were selected for Cg susceptibility. More than ten susceptible plants (putative knockout mutants) have been identified. Open-pollinated seed from each of these susceptible plants was harvested, along with eight resistant siblings as controls.

DNA from a pool of 24 seedlings (grown on paper towels) of each of the putative knockouts and resistant control siblings was extracted. This DNA was used as a template to amplify the flanking sequence from the Mu-gene insertion site using specific primers in combination with a consensus primer designed from the terminal inverted repeat (TIR) from the Mutator element sequence (SEQ ID NO: 125). In other words, PCR products would only be observed if a Mu element had inserted into the candidate gene isolated in Example 2. Primers FLP110F, FLP110R, FLP111F, FLP111R, FLP112F, FLP112R, FLP113F and FLPA1R were used as gene-specific primers (Table 1). The amplified PCR products were blotted onto nylon membranes and hybridized with a DNA probe and the candidate gene isolated in Example 2. PCR products that showed strong hybridization were excised from the gel, purified, cloned and sequenced. The resulting sequences were analyzed by alignment with candidate gene sequences and Mu-TIR elements. Mutator elements cause a direct 9 bp duplication at the insertion site. Based on flanking sequence information and a 9-bp direct duplication, four independent insertions were identified in exon 1 of the candidate gene (Figure 5). An insertion (M177) was detected about 97 bp upstream of the initiation codon, in the 5' untranslated region of the gene. A common case of insertion, 270 bp downstream of the initiation codon, was detected in three susceptible plants: ml64, ml59, and ml79. The susceptible plant M171 was found to contain two Mu- insertions, 556 bp and 286 bp downstream of the initiation codon. When Southern blots were performed with the exon1 region of the gene as a DNA probe, the pattern observed by hybridization confirmed these results.

This and the previous examples can be summarized as follows. Early work cited in Example 1 showed that the previously observed locus conferring Cg resistance was located on the long arm of maize chromosome 4. The nature of this locus, its exact location or the gene(s) encoded by it were completely unknown. The work done in Example 1 demonstrates that the locus can be mapped to a very small region of the long arm of chromosome 4. Example 2 demonstrates that there is a gene to be found in this chromosomal region encoding a new form of an NBS-LRR protein, a family of proteins known to be involved in resistance to pathogens, but which vary greatly in their sequence and resistance specificity. Example 3 shows that this gene is present only in the resistant lineage, and not in the isogenic susceptible lineage, and that the transcripts corresponding to this gene are found in the resistance lineage, indicating that the gene is expressed, and these transcripts are found only in the lineage resistant. Example 4 demonstrates that, in four independently isolated Mu insertion events, when the gene is interrupted by the insertion of a Mu element, the phenotype of the plants is changed from resistant to susceptible to Cg. Taken together, these data provide evidence that the subject matter of embodiments of the present invention is a gene necessary to improve or confer resistance to Cg in maize plants.

Example 5

Backcrossing the Rcgl locus into sensitive lines An introgression of the Rcgl locus from a line was made to confirm that the Rcgl locus could be successfully backcrossed into inbred lines, and that hybrids produced from the inbred line with the Rcgl locus would have improved or attributed resistance. to Cg. DE811ASR (BC5) was also developed and used as an improved donor source for introgression of the Rcgl locus. Next, several additional lines were used as periodic parents in order to utilize the marker-assisted breeding methods described here to efficiently introgress the Rcgl locus into a variety of lines and hybrid genetic backgrounds, reinforcing or conferring resistance to Cg. Each of these examples is discussed in more detail below.

Proof of Concept (PH09B) MP305 is a pure white kernel strain with a strong resistance to Cg, but its final flowering yield, poor agronomic characteristics and weak make it a poor parental donor in the absence of the use of animal-assisted breeding methods. markers described here. A molecular marker profile of MP305 is provided in Table 5b. Primers used for the SSRs reported in the table can be constructed from publicly available sequences found in the Maize GDB on the World Wide Web at maizegdb.org (sponsored by the USDA Agricultural Research Service), in Sharopova et al. (Plant Mol. Biol. 48(5-6):463-481), and/or in Lee et al. (Plant Mol. Biol. 48 (5-6), 453-461). UMC15a is an RFLP marker, and the reported score is based on EcoRl restriction.

To demonstrate the phenotypic value of the Rcgl locus, the locus was first introgressed into PH09B lines (US Patent 5,859,354) to the BC3 stage as follows. The F1 population derived from the cross between MP305 and strain PH09B was backcrossed once again with strain PH09B, resulting in a BC1 population. Seedlings were planted outside and backcrossed back to the PH09B line to develop a BC2 population. DNA was prepared from leaf sacs of BC2 families. To determine BC2 families for plants to backcross, genotyping was performed on DNA from BC2 families using the primers for markers flanking the region of interest, UMC2041, PHI093, and CSU166 (See Table 1). Seeds from BC2 families were planted and individual plants were genotyped for the presence of the MP305 version of this region of the chromosome using the same three markers noted above. Positive plants were backcrossed to the PH09B line once again to develop BC3 populations. Seed from these BC3 populations was planted and the plants were selfed to obtain BC3S1 families segregating for the region of interest, as well as BC3S1 families lacking the region of interest. These families were used for phenotypic comparison (BC3S1 segregating versus BC3S1 without the region of interest).

In order to observe the performance of the Rcgl gene in a heterozygous situation such as would be found in a commercial hybrid, appropriate test crosses were made. Specifically, BC3S1 families segregating to the region of interest were planted and individual BC3S1 plants were genotyped. Plants homozygous for the Rcgl gene as well as homozygous for the null allele (which lacks the gene on both chromosomes) within each family were used to make test crosses with lines PH2EJ (US Patent 6,333,453), PH2NO (US Patent 6,124. 533), PH4CV (US Patent 6,897,363) and PH8CW (US Patent 6,784,349).

In the case of both BC3S1 lines and hybrids, the observed phenotypic differences indicated significant improvement for ASR resistance in lines and hybrids containing the region carrying Rcgl. The effect of the introgressed Rcgl locus in the BC3S1 families and the hybrids derived from the test crosses resulted in an improvement in terms of the number of infected internodes and the number of infected internodes by more than 75%. The results, using a visual accounting system commonly used by plant breeders, are presented in Table 5 below. The data clearly demonstrate that the use of breeding techniques to move the gene from embodiments into other lines genetically competent to use the gene results in greater resistance to Cg. Table 5a: Effect of the introgressed Rcgl region on the degree of resistance to anthracnose, stem rot in BC3S1 families and derived test crosses. Table 5b: Molecular marker profile of MP305 DE811ASR (BC5) like most donors improve its use in backcrosses

Although MP305 was used in the experiment above, 5 as illustrated in Figure 8 (a), DE811ASR (BC5) maintains a smaller MP305 chromosomal interval with the Rcgl locus than DE811ASR (BC3) (and of course MP305 as well), and therefore is particularly useful as a source of donors for the Rcgl gene. The reduced chromosomal interval of DE811ASR (BC5) has been shown to be associated with an improved agronomic phenotype. Twenty-two plants of derived lines DE811ASR (BC3), 20 plants of derived lines DE811ASR (BC5), five DE811 plants and five MP305 plants were grown in a greenhouse from November 2005 to March 2006 and plant height data were collected and ear height; dates when 50% of the plants dropped pollen (midshed), when 50% of the plants had visual ears (midves) and when 50% of the plants had silks protruding from the ears (midslk) and grain coloration was observed. On average, lineage DE811ASR (BC5) was smaller than DE811ASR (BC3) (293 cm vs 345 cm) and ear location was smaller in DE811ASR (BC5) than DE811ASR (BC3) (146 cm vs 183 cm). , both of which are positive characteristics in terms of developing elite varieties. DE811ASR (BC5) was earlier for midshed, midves and midslk compared to DE811ASR (BC3). Midshed was approximately 1 day earlier, midves was approximately 6 days earlier, and midslk was approximately 3 days earlier for DE811ASR (BC5) compared to DE811ASR (BC3). Grains of DE811ASR (BC5) had a brownish-yellow (bronze) color while grains of DE811ASR (BC3) had a pale yellow color. Dates for midshed, midves and midslk were similar for DE811ASR (BC5) and DE811, while MP305 was approximately 11 days after midshed and did not produce 50% visual ears, nor 50% silks during the growing period. Although these data are based on only a few plants for DE811 and MP305 and ears were not produced in these few lines, these greenhouse results resemble observations of these lines in the field. These data indicate that DE811ASR (BC5) resembles the periodic parent DE811 much more closely than DE811ASR (BC3). Thus, DE811ASR (BC5) is an excellent source of starting donors for the Rcgl locus and the Rcgl gene, both genotypically and phenotypically. Furthermore, DE811ASR (BC5) is particularly useful when introgressing the Rcgl locus into germplasm with adaptation similar to DE811. DE811 was developed by J. Hawk (Falcão, JA (1985). Crop Science Vol. 25: p716) and has been described as a yellow inbred line originating from selfing and selection for six generations in a genealogy program crossing B68 to a pure derivative [B37 Ht X (C103 X Mp3204 double crossover) Sci.]. DE811 selected 1 to 2 days later than B73 in trials in Delaware, but 4 days later than B73 in Missouri. Limited yield trials indicate that DE811 has satisfactory compounding ability. It is a good former (good forms, a component of the female corn flower important for fertility) and pollen and can be crossed to recently maturing germplasm for Southern US adaptation and late maturing germplasm for Southern US adaptation. Thus, DE811ASR (BC5), in association with the markers and breeding methods disclosed herein, is useful as a source of initial donors to introgress the Rcgl gene into a wide variety of germplasm, including germplasm adapted to all regions of the U.S. where Cg is present.

Creation of Pure Rcgl Locus Conversions Following the assays for successful introgression of the Rcgl locus into PH09B described above, additional Rcgl locus conversions were performed in other lineages. The first series had backcrosses, with MP305 and DE811ASR (BC5) as donors. For the second series of backcrosses, molecular markers were used to reduce the chromosome gap in the BC5 conversions from the first series. These BC5 conversions were selected for crosses downstream of the Rcgl gene. These selected plants were then backcrossed to create the BC6 generation. Plants with crosses above the gene were selected in the BC6 generation.

First series of backcrosses

In the first series, DE811ASR (BC5) was used as the primary donor source, but parallel introgressions were also made at the same time using MP3 05 as the donor source. These data, described in more detail below, show that, while DE811ASR (BC5) is the preferred donor in many situations, MP305 can also be used effectively with the marker-assisted reproduction methods of the embodiments taught here.

Elite lines primarily adapted to North American growing conditions were selected for use as recurrent parents. The inbred lines initially selected for use as recurrent progenitors were lines PH0R8 (US 6,717,036), PH7CH (US 6,730,835), PH705 (US 6,903.25), PH5W4 (US 6,717,040), PH51K (US 6,881. 881) and PH87P (US 6,888,051). Each of these lines was crossed with DE811ASR (BC5) as well as with MP305. The F1 generation derived from each of these crosses was backcrossed once again with the respective inbred line, resulting in a first backcross generation (the recurrent parental BC1). Seedlings were planted outside and DNA was prepared from leaf bags. PCR reactions were performed using primers for markers flanking the region of interest; UMC1466, UMC1418, BNLG2162, UMC1086, UMC2041, UMC1612, CSU166, UMC1051, UMC2187, UMC1371 and UMC1856 were used in the first rounds BC (See Table 1), while in the later rounds BC, UMC1418, BNLG2162, UMC1051, 1, UMC2187, UMC1371 and UMC1856 were used. Seedlings whose PCR reactions gave a positive result (meaning that MP305 derived from the Rcgl locus was present) were then further backcrossed to the respective inbred lines to make a BC2. This procedure, called "genotyping," identifies the genetic makeup of a plant at the location of a specific marker. These measurements were repeated for the recurrently developing parental BC3, BC4, and BC5. Analysis shows that after five backcrosses, these lines maintained a significantly truncated chromosomal interval comprising the Rcgl locus and, based on visual observations, no indication of negative effects resulting from the presence of the Rcgl locus was observed.

Recurrent parental selection was also performed, selecting the most phenotypically similar plants as the recurrent parent. Using these methods genotypically and phenotypically, high-quality conversions were selected with a high percentage of recurrent parental across the entire genome.

This example also illustrates that flanking markers are not used exclusively to select for or away from the Rcgl gene. Seedlings whose PCR reactions gave a positive result (meaning that the MP305 derivative of the Rcgl locus was present) were then further backcrossed to the respective inbred lines to make the final backcross (the recurrent parental BC5 generation) in this first series. Where the closest flanking polymorphic markers determined that the gene was present, the next set of double polymorphic markers most distally flanking the gene were used for recurrent parental selection. Thus, the use of markers flanking the Rcgl gene or Rcgl locus serves to illuminate the recombination occurring in the region.

Second series of backcrosses

Line conversions of the Rcgl locus made using the SSRs flanking the Rcgl locus in the first series of backcrosses were then used as donors in a successive round of backcrossing. For this series of backcrosses, SNP markers were developed for the Rcgl gene that allowed assisted selection in a high-throughput manner, as described in Example 15, to select for the Rcgl gene. SNP markers were also designed in the region around the Rcgl locus, allowing flanking markers to be used to select away from the MP305 chromosomal interval around the Rcgl locus and select for the recurrent parental genotype, significantly reducing linkage drag. It is only through physical mapping and gene cloning that such marker-assisted recurrent parental selection is possible.

First, the BC5 recurrent parental plants resulting from the first series of backcrosses were reselected with the most accurate marker set, and recombination was selected south of the Rcgl gene. Flanking markers strongly linked to the Rcgl gene (MZA8761, MZA1851, UMC1051, and UMC2187) were used to select for the recurrent progenitor south of the gene in small population sizes of about 40 offspring. (See figure 8 (ab)). These progenies were then analyzed using the FLP markers disclosed herein to more precisely determine the point of recombination. These data showed that some offspring were selected with the recurrent parental genome less than 1 cM (based on maps of genetic distances between IBM2 neighbors) south of the Rcgl gene, as shown in Figure 8(b). Other progeny had a recurrent parental genome less than 4 cM south of the Rcgl gene. These marker-selected BC5 conversions were then used as donors and crossed to near-isogenic counterparts of PH705, PH5W4, PH51K, and PH87P as the periodic parents to give a BC6 population. Markers in the Rcgl gene were again used to select for Rcgl, with flanking markers to the north this time of Rcgl being used to select for the recurrent parental. In this round of selections, recombinations were detected in each population between Rcgl and the MZA15842 marker. The position of MZA15842 on the genetic map of IBM2 neighbors can be extrapolated from its position on the high-resolution map shown in Figure 7(b), map B, by regression against the flanking markers UMC2285 and PHI093. This MZA15842 was placed at 520.5 cM on the genetic map of IBM2 neighbors. Therefore, as shown in Figure 8(b), in two cycles of backcrosses, the donor genome was reduced to a segment of less than 6 cM in each population, or less than 0.8% of chromosome 4, based on distances. from the genetic map of IBM2 neighbors, and in some descendants the segment was smaller than 2.1 cM, or less than 0.25% of chromosome 4. For comparison purposes, the MP305 chromosomal interval with the Rcgl locus in DE811ASR (BC3 ) was 131 cM, or approximately 16% of chromosome 4, based on genetic map distances from IBM2 neighbors. It is only through physical mapping and cloning of the marker gene that accurate and efficient assisted selection, with recurrent parental, is possible.

A more in-depth analysis

Therefore, as a result of good mapping of the location of the Rcgl gene, anyone can use two flanking markers that are genetically linked with the Rcgl gene to select a small chromosomal region with crossovers north and south of the Rcgl gene. This has the benefit of reducing linkage drag, which can be a confounding factor when trying to introgress a specific gene from adapted germplasm, such as MP305, into elite germplasm, such as the lines mentioned above. Figures 7 and 22, and Table 16 show many combinations of markers flanking the Rcgl gene and locus that can be used for this purpose. Some specific flanking markers that can be used for selection of truncated chromosomal intervals that include the Rcgl gene or locus are UMC2285 and UMC15a, UMC2285 and UMC2187, UMC1086 and UMC2200, UMC2041 and UMC2200, UMC2041 and PHI093, MZA11455 and UMC15a, 455 and MZA3434, MZA15842 and MZA3434 and FLP8 and FLP33. Optionally, on or within each of these chromosomal intervals, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more markers in order to locate the recombination event and choose for the Rcgl gene or Rcgl locus with the maximum amount of the recurrent parental genotype. Additionally, one can have at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more markers between the northern end of the chromosomal interval, as well as from the top of the Rcgl gene and/or between the southern end of the chromosomal interval and the bottom of the Rcgl gene.

It is advantageous to have closely linked flanking markers for selection of a gene, and highly advantageous to have markers within the gene itself. This is an improvement over the use of a single marker or distant flanking markers, since with a single marker or with distant flanking markers the link associated with Rcgl can be broken, and by a selection of such markers it is more likely that , inadvertently select plants without the Rcgl gene. Since marker-assisted selection is often used instead of phenotypic selection, a marker-trait association has been confirmed, the unfortunate result of such an error would be to select plants that are not resistant to Cg and discard plants that are resistant to Cg. Cg. In this sense, markers from within the Rcgl gene are particularly useful, as these, by definition, remain linked to Cg resistance as enhanced or conferred by the gene. Furthermore, markers from within the Rcgl locus are so useful for a similar reason. Due to its close proximity to the Rcgl gene, it is highly likely that it remains linked to the Rcgl gene. When introgressed with the Rcgl gene, such elite lines can be used both for the production of hybrid seeds and as a source of donors for further introgression of the Rcgl gene into other lines.

Thus, the data clearly show that inbred offspring converted using DE811ASR (BC5) as a donor source retain the truncated MP305 chromosome interval. The strains that make up the truncated MP305 chromosomal interval are very useful as donor sources in their own right, and there is no need to revert to DE811ASR (BC5) as a donor source. Using marker-assisted reproduction as described here, the truncated MP305 chromosomal interval can be further reduced in size, if necessary, without concern of losing the link between the markers and the Rcgl gene. Phenotypically, a reduced chromosomal interval is associated with better agronomic performance, as demonstrated by DE811ASR (BC5) versus DE811ASR (BC3), described above.

Example 6

Constructs for transgenic expression of Rcgl The Rcgl gene was expressed as a transgene to determine whether this gene alone was sufficient, as well as necessary, for the phenotype conferred by the Rcgl locus. This was done using two different constructs, differing mainly in the 10 promoter used.

Example 6a:

In this example, the Rcgl gene was expressed using its own promoter. The upstream region of the Rcgl gene was sequenced using BACs as in Example 2, which provided the sequences of the protein-coding section of the gene. The 1684 bp 5' sequence to ATG is set out in SEQ ID NO: 24.

In order to transform the complete Rcgl gene, including the promoter and protein coding region, a 5910 bp fragment extending from position 41268 to position 47176 in SEQ ID NO: 137 was amplified by PCR using a clone BAC as a DNA model. To allow cloning using the Gateway ® cloning system (Invitrogen, Carlsbad, USA), ATTB 25 sites were incorporated into the PCR primers, and the amplified product was cloned into the pDONR221 vector by GATEWAY ® BP recombination reaction. The resulting fragment, flanked by attL sites, was moved by the GATEWAY ® LR recombination reaction into a binary vector. The DNA construct was then used to transform corn, as described in Example 7.

Example 6b:

In order to express the Rcgl gene throughout the plant at a high level, the coding region of the gene and its terminator were placed behind the promoter, in the 5' untranslated region and an intron of a maize ubiquitin gene (Christensen et al. (1989) Plant Mol. Biol. 12:619-632; Christensen et al. (1992) Plant Mol. Biol. 18:675-689). One skilled in the art would be able to select a different promoter to express the Rcgl gene based on the desired expression pattern. To allow cloning using the Gateway ® cloning system, ATTB sites were incorporated into the PCR primers that were used to amplify the Rcgl gene from 142 bp upstream of the start codon and 624 bp downstream of the stop codon. The amplified product was cloned into pDONR221 (Invitrogen, Carlsbad, USA) using a Gateway ® BP recombination reaction. After cloning, the resulting Rcgl gene was flanked by attL sites. In the final step, the Rcgl clone was moved by the GATEWAY ® LR recombination reaction into a vector containing the maize ubiquitin promoter in the 5' untranslated region and first intron of the ubiquitin gene as described by Christensen et al. (supra), followed by GATEWAY ® ATTR1 and R2 sites for insertion of the Rcgl gene behind the ubiquitin expression cassette. The vector also contained a marker gene suitable for maize transformation, so that the resulting plasmid carrying the chimeric gene (maize ubiquitin promoter - ubiquitin 5' untranslated region - ubiquitin intron 1 - Rcgl) was suitable for maize transformation as described in Example 7.

Example 7

Agrobacterium-mediated maize transformation and transgenic plant regeneration

The recombinant DNA constructs prepared in Examples 6a and 6b were used to prepare the transgenic maize plants as follows. Corn was transformed with selected polynucleotide constructs described in Example 6a and 6b using the method of Zhao (US Patent No. 5,981,840, and PCT patent publication WO98/32326). Briefly, immature embryos were isolated from corn and the embryos contacted with a suspension of Agrobacterium, where the bacteria were able to transfer the polynucleotide construct into at least one cell of at least one of the immature embryos (step 1: infection stage). . At this stage, the immature embryos were immersed in an Agrobacterium suspension to begin inoculation. The embryos were co-cultured for a while with Agrobacterium (step 2: the co-cultivation step). Immature embryos were cultured on solid medium after the infection stage. Following this period of co-cultivation an optional "rest" stage was carried out. In this resting stage, the embryos were incubated in the presence of at least one antibiotic that inhibits the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting stage). Immature embryos were cultured on solid medium with antibiotics, but without a selection agent, to eliminate Agrobacterium. and a resting phase for infected cells. Then, the inoculated embryos were cultured in medium containing a selective agent, and callus transformed into growth was recovered (step 4: selection step). The callus was then regenerated into plants (step 5: regeneration step) and calli grown on selective medium were grown on solid medium to regenerate the plants.

Example 8: The Rcgl gene is necessary but not sufficient to confer the phenotype associated with the Rcgl locus

Transgenic plants were made as described in example 7 using the constructs described in examples 6a and 6b, respectively. For the native Rcgl gene and the Rcgl ubiquitin gene constructs, 30 independent events and 10 control events transformed with the binary vector alone, without the constructs of Example 6a or 6b, were generated.

Leaf discs from each native gene transgenic event were harvested for total RNA isolation. RT-PCR was performed using primers specific to the FLP111F gene and set forth in SEQ ID NOS: 37 and 38. In 29 of the 30 transgenic events, the expected 637 RT-bp PCR bands were present, indicating expression of the gene construct. native. Disease assays were performed in the greenhouse on the same 30 native Rcgl transgenic events to determine whether the plants were resistant to Cg. To this end, leaf rust assays were the first to be performed on 5 sister plants from each event using the procedures described in Example 12. A single event was found to show a statistically significant reduction in disease score relative to control plants that lost the native Rcgl gene construct. Plants that had been subjected to the leaf burn test were allowed to develop two weeks after anthesis and were then further tested by inoculating Cg into the first elongated internode of the stem. These stem infection assays showed a single transgenic event expressing the native Rcgl transgene to be more resistant to Cg infection relative to control plants. However, this event differed from the positive event identified through foliar infection assays.

Plants transformed with the Rcgl ubiquitin construct described in Example 6b were analyzed similarly. RT-PCR showed that 27 of the 30 transgenic events contained the expected 637 bp bands, indicating expression of the Rcgl ubiquitin construct. When foliar infection tests were performed on 5 plants from each of the 30 events, a single event was identified that showed a statistically significant reduction in plant disease control. The transgenic plants were analyzed by stem infection tests. Two events were found to present greater resistance to stem rot when compared to control plants that lost the Rcgl ubiquitin gene. These transgenic events do not include the positive event identified in the leaf spot assays. An Rcgl ubiquitin event showed a statistically significant increase in susceptibility to anthracnose stem rot, relative to control plants.

The results of these experiments suggest a lack of efficacy with either native or ubiquitin Rcgl constructs in leaf blight or stem infection assays. Although by RT-PCR, each construct was found to be expressed at levels comparable to or greater than the level of Rcgl found in DE811ASR (BC5), only a limited number of events were found demonstrating a statistically significant improvement in disease score relative to to control plants: 2 native TO Rcgl events and 2 ubiquitin TO Rcgl events. These events were identified separately by leaf burn and stem infection assays. However, positive events showing improved disease resistance by the leaf burn assay do not correlate with those identified by the stem infection assay. This is in contrast to the positive control DE811ASR (BC5), which shows a clear increase in relative resistance to DE811 in both the leaf scorch and stem infection tests. Furthermore, no correlation was observed between the measured disease scores and the level of transgene expression. Indeed, one of the ubiquitin-positive Rcgl events failed to produce a detectable RT-PCR band upon reverse transcription of total RNA and amplification of DNA with FLP111F and FLP111R. The lack of correlation between the different assays for the small number of events that appeared to show resistance, the larger number of events that did not show resistance, and the very different results obtained with DE811 (BC5) led to the hypothesis that while the Rcgl gene is necessary for the phenotype conferred by the Rcgl locus, it alone is not sufficient. The small amount of apparent resistance observed is due to normal experimental variation.

In order to test the aforementioned hypothesis, and exclude the possibility that errors inherent in the Rcgl coding region of both constructs give rise to the production of a non-functional protein that is not competent in the expression of resistance, an experiment was developed who tested the ability of transgenes to complete a transposon knockout in Rcgl. The ml64 insertion line, identified in MP305 of the Mu- target population and described in Example 4, contains a transposable element in exon 1 of the anthracnose Rcgl gene and exhibits a phenotype susceptible to Cg infection. This lineage therefore has all components of the Rcgl locus, but the Rcgl gene is not functional due to transposon insertion. Two native strains and two Rcgl TI ubiquitin strains (i.e., two of each of the strains carrying the constructs of Examples 6a and 6b, respectively) were crossed with the Rcgl ml64 mutant strain. Descendants of the cross were selected by painting the leaf with glufosinate to identify plants containing the selection marker gene that co-segregates with the Rcgl transgenes. Plants carrying the target Rcgl Mu gene were identified by PCR of extracted DNA using an Rcgl gene-specific primer defined in SEQ ID NO: 259 and the Mu-TIR primer of SEQ ID NO: 233. Leaf sheaths of V7-9 plants were inoculated with 5 □! of a suspension of 5 x 10 6 / mL of C. graminicola conidia after tissue wounding. Inoculated leaf sheaths were sealed in plastic for 5 days to maintain high humidity levels. At this time, the most infected sheath was excised to quantify the size of the lesion by LemnaTec image analysis (LemnaTec, Aachen, Germany).

Data from the leaf sheath infection assays are summarized in Figure 33, and a typical result of an experiment is shown in Figure 27. In the absence of the native Rcgl transgene, leaf sheaths of plants containing the Rcgl Mu-target gene showed great expansion of the necrotic lesions that typify the reaction of this susceptible ASR plant. In contrast, when the native Rcgl transgene is present, an ASR-resistant phenotype is restored and necrosis is confined or restricted to the site of wounding and fungal inoculation. The data in Figure 33 show that the area of necrosis decreases from an average of 0.76 cm 2' in

Similarly, lesion size in plants containing the Rcgl gene target transposon decreases from an average value of 0.89 cm2 in the absence of the Rcgl ubiquitin transgene to 0.11 cm2 in the presence of the Rcgl ubiquitin transgene.

Thus, both the native and Rcgl ubiquitin transgenes are capable of completing the knockout mutation in Rcgl and restoring an ASR-resistant phenotype, indicating that both constructs are functional. The fact that no transgenic construct alone is capable of offering resistance indicates that the Rcgl gene is not sufficient for stem rot resistance. The Rcgl locus therefore contains an additional component that is required for Cg resistance. The Rcgl gene is necessary but not sufficient.

Example 9

Analysis of DE811ASR BAC Clone Sequences to Identify ASR Resistance Components The BAC clones from Example 2 covering the Rcgl introgressed region of DE811 ASR were examined for potential candidate genes that may be required in addition to the Rcgl resistance gene for root rot. thatch. The sequences of four overlapping BAC clones were inspected to identify predicted genes with homology to putative disease response related proteins. A 79 kb supercontig (stretch of 79 kb ordered, nearly continuous sequence, designated H supercontig) in 191 BAC clones was found to contain sequences encoding a protein showing homology to multiple proteins

NBS-LRR from barley and rice. The alignment is shown in Figures 31a through 31g. This segment of the contiguous BAC covered by 4101 bp, contained 2 predicted exons, an intron and 1164 encoded ORF (open reading frame) amino acids. Sequence comparison against a database of protein signatures revealed the presence of a typical LRR domain, an NB domain with subdomains, and a short protein kinase-like sequence in the last 37 amino acids of the ORF. In order to determine whether the NBS-LRR sequence was encoded by a functional gene that was expressed in DE811ASR plants, RT-PCR was performed. Total RNA (750 ng) from uninoculated and Cg-inoculated DE811 and DE811ASR samples (3,6,9,13 DPI) was copied into DNA using Reverse Transcriptase SUPERSCRIPT ® III (Invitrogen, Carlsbad, California, U.S.A.) and amplified using PLATINA ® Taq DNA polymerase (Invitrogen, Carlsbad, California, USA). The DNA synthesis reaction was set up according to the protocol provided by the manufacturer, using random hexamers as primers and incubated at 50 □ C for 50 minutes. The reaction was then terminated by heating at 85°C for 5 minutes and the RNA template was removed by RNaseH digestion at 37°C for 20 minutes. Primer pairs were selected to amplify 3 different NBS-LRR template cDNA fragments.

These PCR primer pairs consisted of ETJ111 (SEQ ID NO: 234) and ETJ112 (SEQ ID NO: 235); ETJ113 (SEQ ID NO: 236) and ETJ114 (SEQ ID NO: 237); ETJ115 (SEQ ID NO: 238) and ETJ116 (SEQ ID NO: 239). cDNA (1 □ L reverse transcriptase reaction) was amplified for 30 cycles consisting of 30 seconds at 94°C, 30 seconds at 50°C, and 1 minute at 72°C, followed by a 7 minute extension at 72°C. °C. Figure 28 shows the results of the RT-PCR performed with the primer pair ETJ115 - ETJ116. This data clearly shows that the NBS-LRR gene from the BAC clone of the 191 H supercontig is expressed exclusively in DE811ASR plants that contain the introgressed MP3 05 region. Similar results were obtained when DNA amplification was performed using primer pairs ETJ111-112 and ETJ113-114. DNA sequence analyzes found that all three RT-PCR products were derived from the NBS-LRR sequence of the H supercontig. from BAC191.

The NBS-LRR sequence obtained from the BAC clone does not contain an obvious stop codon and was therefore only partial in nature. In order to define the end of the coding region and obtain the complete transcript sequence, a 3' RACE experiment was performed. Total RNA from uninoculated DE811ASR and Cg inoculated (6DPI) plants was copied into DNA using SUPERSCRIPT ® III RT and an oligodT primer using standard conditions. The DNA was then subjected to two cycles of amplification with Platinum ® Taq DNA polymerase using gene-specific primers OETJ118 (SEQ ID NO: 241) and OETJ119 (SEQ ID NO: 242) and an oligodT adapter primer, oETJ117 (SEQ ID NO : 240). The results of this procedure predicted an additional 976 bp of sequence. Combination of the sequences from the BAC 191 clone and the RACE 3' product led to the prediction of an approximate 4.5 kb transcript encoding a 1428 amino acid polypeptide. In order to confirm this composite sequence, a full-length cDNA was isolated by RT-PCR using SUPERSCRIPT ® III RT, in combination with Phusion ™ Hot Start High Fidelity DNA Polymerase (New England Biolabs, Ipswich, Massachusetts, USA). cDNA synthesis was prepared by oligodT using total RNA from DE811ASR plants (0.13 DPI). Amplification with Phusion™ polymerase employed gene-specific primers oETJ 120 (SEQ ID NO: 243) and OETJ121 (SEQ ID NO: 244) that were positioned to amplify a fragment extending from the translational start site at 31 bp to the stop codon. Sequence analysis of the resulting 4318 bp fragment showed perfect agreement between the cDNA sequence and the BAC composite clone plus the RACE sequence product.

The sequence of the 4318 bp cDNA fragment is given in SEQ ID NO: 245. The deduced 1428 amino acid sequence of the 4287 bp ORF is set forth in SEQ ID NO: 246. The gene was designated Rcglb. BLAST P ® homology search performed on the 1428 aa sequence revealed good homology of the NBS-LRR sequences throughout the first ~ 900 amino acids of the sequence (Figures 31a through 31g). Furthermore, good homology was found between amino acids 1036-1399 of the sequence (set forth in SEQ ID NO: 256) and several putative protein kinases (Figures 32a and 32b). When the protein sequence is compared against a database of protein families, domains and sites, a tripartite structure consisting of an NB-ARC, LRR and protein kinase domain is obtained. The domain structure of the Rcglb polypeptide is diagrammed in Figure 29. The inclusion of these three domains within a single polypeptide is not commonly found. However, a barley NBS-LRR-PK gene, Rpg5, proposed that the function of resistance to Puccinia graminis has been identified (Brueggeman et al., (2005) Abstracts of Plant and Animal Genomes XIII Conference, Poster 320 [online], [visited on 06-08-2007]. Retrieved from the Internet: <URL: htt p: / intl- pag.org/13/abstracts/PAG13 P320.html />.

Comparison of the cDNA sequence with the BAC clone genomic sequence indicates that the Rcglb gene is divided into 9 exons and 8 introns and spans more than 44 kb. Introns 2-8 are concentrated in the protein kinase portion of the gene. Two of these introns are extremely long: intron 2 is 16.7 kb and intron 5 is 20.6 kb. The initial genome sequence that was obtained for the Rcglb gene and its flanking regions are provided in SEQ ID NOs:255-258. Due to the highly repetitive nature of some of the sequence elements between the two genes, some areas of the genomic DNA presented significant sequencing difficulties. After initial sequencing, there were three sequence gaps located in intron 5 of the Rcglb genomic sequence. The segment of the genome sequence corresponding to the first 5 exons and first 4 entire introns, as well as the beginning of the 5th intron (before the first sequence gap) is established in SEQ ID NO: 255. This segment includes the initiation codon (ATG ), the TATA box, and a CAAT element. The second and third elements of the Rcglb genomic sequence correspond to two portions of intron 5, between which the second sequence gap exists, and are set forth in SEQ ID NOs: 256 and 257. The fourth and final segment of the Rcglb genomic sequence Rcglb corresponds to the remainder of intron 5, after the third sequence gap, through the remaining 3 total introns and 4 complete exons. This segment is set forth in SEQ ID NO: 258, and includes the stop codon of the Rcglb sequence, as well as the 3' UTR and 3' flanking genomic sequences. Further sequencing efforts were able to eliminate the intron 5 gaps from the genome sequence for Rcglb. The complete genome sequence for the Rcglb gene is therefore set forth in SEQ ID NO: 266, with the positions of all exons and introns indicated in the sequence accompanying the present application. There is a small region within SEQ ID NO: 266 represented by a stretch of 21 "n" residues that is not a gap, but is a result of poor sequence quality in which the 21 positions have not yet been positively confirmed. Currently, the complete DNA sequence of the genomic interval between Rcgl and Rcglb is not known because there is a small remaining gap in the genomic DNA sequence located between the two genes, which has been difficult to sequence due to repetitive elements.

Northern blot analysis allowed independent confirmation of Rcglb expression and provided an estimate of transcript size. Northern blots of polyA + RNA (0.7 ug) from resistant and susceptible plants (0, 6, 13 DPI) showed the presence of a 4.75 kb transcription band, present only in resistant samples that hybridized with specifically prepared probes from LRR and the kinase region of the gene. (Figure 30). The observed transcript size agrees with the size calculated from the cDNA and RACE products (4.68 kb) An MPSS ® tag predicted from the Rcglb cDNA sequence is found to be present at a low level (<144 ppm) , only in DE811ASR libraries. This finding is consistent with RT-PCR and Northern blot results and confirms the exclusive presence of this low transcript abundance in ASR-resistant plants.

Example 10

Dual expression of Rcgl and Rcglb as transgenes; Both Rcgl and Rcglb transgenes are required for resistance to Colletotrichum graminicola

The Rcgl and Rcglb genes can be expressed as transgenes, allowing the modulation of their expression in different circumstances. The following disclosure shows how the two genes can be expressed in different ways to combat different diseases or to protect different parts of the plant, or simply to move the two genes into different lines of corn as transgenes, as an alternative to moving the locus Rcgl all through the crossover, as described in Example 5.

To assemble an Rcglb gene driven by its native promoter, 3 Rcglb fragments were joined into a GATEWAY ® entry vector containing attL3 and attL4 recombination sites, separated by a multiple cloning site region. A 2.4 kb fragment of BamHI-Kpnl containing 1967 bp of the downstream sequence and 432 bp of the Rcglb coding region, a 3.7 kb Kpnl fragment of the Rcglb cDNA, and a 1.1 kb BSAI fragment of the Rcglb cDNA. SmaI containing 146 bp of the Rcglb cDNA plus 93 9 bp of 3' flanking DNA were joined into a 4-linkage of BamHI-SmaI digest of the L3/L4 entry vector to generate Construct A. The native promoter of the Rcglb construct was driven was then transferred by recombination along with the native Rcgl constructs from Example 6a into a destination vector containing attLl/attL2 and attL3/attL4 sites to give a stacked Rcgl-promoter-Rcgl/Rcglb-promoter-Rcglbl construct (Construct B).

For high level constitutive expression, a ubiquitin promoter-directed Rcglb construct was generated. For this construct, an Rcglb fragment reaching from 1 to 4318 nucleotides of the cDNA sequence was amplified, a BamHI-SnaBI fragment by RT-PCR and inserted into a GATEWAY ® entry vector containing the ubiquitin promoter, 5'UTR, intron and the Pinll terminator flanked by attL3 and attL4 sites to produce Construct C. An analogous construct was generated for Rcgl (Construct D), in this case a GATEWAY ® entry vector, where the ubiquitin promoter and Pinll terminator are accompanied by attLl sites and attL2. Both the Rcglb and Rcgl ubiquitin-directed genes were transferred by an LR recombination reaction into the destination vector before timing this point to give a Vfbi-Rcgl/Ubi-Rcglb stacked construct (Construct E). One skilled in the art would be able to select a different promoter to express the Rcgl and Rcglb genes, or two different promoters, each expressing one of the two genes.

The above-described constructs were transformed into corn using the method of Example 7. Two separate experiments were performed involving transformation of the construct of Example 6a, Construct A, and Construct B in one experiment and Construct C, Construct D, and Construct E in the other. For the first experiment, a total of 15, 10, and 30 events were generated from transformation of corn with the construct of Example 6a, Construct A, and Construct B, respectively. To confirm the expression of the DNA constructs, molecular analyses were performed. Leaf discs from each transgenic event of the native gene were harvested for isolation of total RNA. RT-PCR was performed using the Rcgl-specific primers AlexlF and AlexlR set forth in SEQ ID NOs: 260 and 42, respectively, and the Rcglb-specific primers ETJ144 and EJT145 set forth in SEQ ID NOs: 261 and 262, respectively. After RT-PCR, all 10 events from Construct A contained the expected 491 bp bands of Rcglb and all 15 events transformed with the Construct of Example 6a contained the expected 420 bp bands of Rcgl. Of the 30 events generated from transformation with the native Rcgl and Rcglb genes (Construct B), 29 expressed both transgenes, while a single event was found to express one transgene (Rcgl) but not the other (Rcglb). Disease assays were performed in the greenhouse on the same transgenic events in order to determine whether the plants were resistant to Cg. Leaf sheath assays were first performed on 3 sibling plants from each event using the procedure described in Example 8. Infected plants were scored 5 days after Cg inoculation by quantifying lesion size using LemnaTec image analysis (LemnaTec, Aachen, Germany). No significant difference in lesion size was found between transgenic plants containing any of the 3 different constructs (Table 6). The mean lesion size for plants containing the construct of Example 6a was 1.286 cm2, whereas that for Construct A and Construct B was 1.070 and 1.174 cm2, respectively. Thus, while RT-PCR data indicated that all three constructs showed good expression in transgenic plants, no effect on Cg resistance could be demonstrated for either the Rcgl or the native Rcglb gene construct alone or stacked for the construct of both genes in combination. Similar results were obtained when the same transgenic events were subjected to stem inoculation with Cg. Table 6a: Transgenic plant results: lesion size

In contrast to the results obtained with the native gene constructs, significant differences in Cg resistance were found for the transgenic plants containing the constructs driven by the maize ubiquitin promoter. As shown in Table 6b, the average lesion size for plants containing single gene Construct C (Ubi-Rcglb) and Construct E (Ubi-Rcgl) were 1.879 and 1.897 cm2, respectively. However, plants containing the overlapping genes Ubiquitin Gene Construct (Construct D) showed a significant reduction in lesion area with the corresponding mean lesion size of 0.345 cm2'. These results are shown in Figures 34 to 36. RT-PCR confirmed the expression of the Rcglb transgene in 15/15 events of Construct C and of the Rcgl transgene in 13/15 events of Construct E. Good expression of both Ubiquitin-promoter directed transgenes was found in 27/30 events of Construct D. Two events lost detectable expression of both transgenes; one event showed strong expression of Rcglb but no apparent expression of Rcgl. Data from the leaf sheath infection assay indicate that neither Rcgl nor Rcglb alone confers resistance to Cg and instead both Rcgl and Rcglb are required for expression of the resistance phenotype. Table 6b: Transgenic plant result: lesion size

An interesting and unexpected observation mentioned above is that the Rcgl and Rcglb transgenes, driven by the ubiquitin promoter, together provide resistance to Cg 5 infection while the transgenes driven by its endogenous promoter do not. It is known that the ubiquitin promoter (in combination with intron I of maize ubiquitin) is capable of generating high level of constitutive expression of markers and trait genes in varied tissues under varied environmental and developmental conditions. 10 Detailed characterization of fragments of the Rcgl or Rcglb promoter used in the Constructs of the present invention has not been performed. The native Rcgl gene in the Construct of Example 6a was shown in Example 8 to complete a Muno gene insertion knockout, indicating that the Construct, and thus the included promoter fragment, is functional. Given that no detailed study of the Rcglb promoter fragment of Construct A and Construct B has been performed, it appears that not all information necessary for precise gene regulation is encoded in this 5' fragment. The infection data indicate that when the 1835 bp promoter fragment is used to drive expression, Rcglb is not correctly expressed. Although the functional Rcglb promoter has not yet been defined, the Ubi-Rcglb results demonstrate that any functional promoter can be used to drive gene expression and this can be combined with Rcgl expression to produce resistance to Cg infection.

Example 11

Analysis of the Distribution of the Rcgl Gene through Germplasm and Identification of Rcgl Sequence Variants

After identification, sequencing and fine mapping of Rcgl, other lineages were selected for the Rcgl gene. To determine the presence of the Rcgl gene in maize germplasm, gene-specific primer combinations FLP111F and FLP111R as well as FLP113F and FLPA1R were used to amplify genomic DNA from a diverse panel of maize lines, including the lines listed in Table 18 and F2834T. , by the polymerase chain reaction. In only 14 (including MP305) out of the panel maize lines was an amplification product detected, indicating that the Rcgl gene is present in only a very small percentage of the lines that were selected. Therefore, in addition to using MP305 or DE811ASR (BC5) as a donor source, other sources containing the Rcgl gene can also be used as a donor source. For example, the public TX601 inbred lines (available under National Plant ID 'Ames 22763'

Germplasm System (NPGS)) and F2834T (available at ID 'Ames 27112' from NPGS), which contain the Rcgl gene can be used as donor sources in crossing with other maize lines that do not contain the Rcgl gene and selecting for the gene Rcgl using markers as described here.

Variants of the Rcgl gene have also been identified and analyzed for single nucleotide polymorphisms (SNP). SNPs were identified at positions in Sequence ID number 1 corresponding to one or more of positions 413, 958, 971, 1099, 1154, 1235, 1250, 1308, 1607, 2001, 2598 and 3342. (see Table 7). Not all allelic variants of the Rcgl gene indicated a resistant phenotype. Therefore, these SNPs can be used as markers to accurately identify and control the Rcgl sequence in a plant genetic improvement program, and to distinguish between resistant and susceptible allelic variants. Furthermore, these SNPs indicate that there are variant sequences that show a resistant phenotype and can be used in the methods and products disclosed here. Four other lineages were also found to contain an Rcgl allele: BYD10, 7F11, CML261 and CML277. Tests of 10 plants did not provide sufficient data to conclusively determine whether 7F11 lines are resistant. There are no data available on the resistance of strains BYD10, CML261 and CML277. Table 7: SNPs identified Phenotype in allelic variants of the Rcgl gene Plants Consensus position Tested SEQ ID NO: 1 Resistant from DE811ASR (BC5) PHBTB Resistant 413 958 971 1099 1154 1235 1250 1308 1607 2001 2598 3342 More than 500 AAGCCAACAAGC plants above 4-5 years 150-210, after A A G C 3 years C A A A A G C PH26T Resistant 50, more than 1 A A G C A A year A A G C TX601 Data 10, more than i A A G c C 7 A insufficient year F2834T No data —- A A G c C A A B54 No data —- C C C T A A T PH0RC Data 19, more than i C C C T A A T insufficient year PH277 Data 17, more than i C c C T A A T A A G C A A G C G G A A G G A A G G A A 189 / 222 PHDGP insufficient year Susceptible 150-210, after CCCTAAT : G G A 3 years PHDH7 No MP data 305 publishes Resistant 50 0

Consensus Length = 4212 nucleotides. SEQ ID NO: reads Rcgl sequence. For the remaining lineages, the available sequence extends from the "atg" start codon in the first exon to the "tga" stop codon in the second exon. The consensus position is based on 5 SEQ ID NO: 1. A A C 10

Example 12

Lines containing the Rcgl locus are resistant to anthracnose-induced leaf blight. The closely isogenic lines DE811 and DE811ASR described in Example 1 were tested for differences in resistance to leaf spot caused by Cg using the following procedure. Four common household sewing needles were glued to a metal holder, where the holes for the thread extended outward from the piece of metal, with four needles extending an equal distance. This apparatus was dipped into a Cg spore suspension at 5 x 10 spores/mL and then pushed across the surface of a young corn leaf in such a way that the leaf was wounded and the wounds simultaneously inoculated with the spores. A damp cotton swab was placed over the midrib near the inoculation site and the entire area covered with plastic film and, over this reflective film, both were connected with adhesive tape, to keep them moist and shaded. The plants were left in this state for 50-54 hours in a standard greenhouse, after which the tape, fabric and plastic film were removed. At 7 and 15 days after inoculation, the size of the lesion was measured and recorded in units of square centimeters.

Figure 10 (a-b) shows the distribution of lesion sizes 15 days after inoculation on all individual leaves. Lesion sizes vary in each data set, but virtually all DE811 leaves (Figure 10b) had lesion sizes significantly larger than the largest lesions found in DE811ASR (BC5) leaves (Figure 10a). Data are summarized for both the 7th and 15th post-inoculation days of the data sets in Figure 11. On both the 7th and 15th days, the average lesion size was smaller in leaves carrying the Rcgl gene. The difference becomes greater over time, the fungus has time to grow and cause more damage, so that although the difference is about twice less in 7 days, by 15 days it is more than four times, and by In fact, the fungus has advanced less in the leaves of DE811ASR (BC5). These results clearly demonstrate that the presence of the locus containing the Rcgl gene confers resistance to anthracnose leaf spot.

Example 13

Hybrid lines derived from DE811ASR (BC5) have higher yields than hybrids derived from DE811 when infected with Colletotrichum graminicola.

In order to demonstrate that corn hybrids containing the Rcgl locus have greater yield potential when infected with Cg than the hybrid lines lacking Rcgl, DE811ASR (BC5) and DE811, the isogenic lines described in Example 1 were each crossed to the inbred lines B73Ht and Mol7Ht, which are both susceptible to Cg.

Hybrid lines were grown and evaluated for Cg response in 2005 at six locations in five different U.S. states. For each hybrid line, three replications of four lines were planted at approximately 74,000 plants per hectare. Plants were inoculated with Cg at the base of the stem approximately 10 days after flowering. The first line of each four line plots was evaluated to determine whether inoculations had been successful by determining the response to Cg four to five weeks after inoculation. The stems were divided and disease progression was scored by observing the characteristic black color of the fungus growing up the stem. Disease assessments were performed as described by Jung et al. (1994) Theoretical and Applied Genetics, 89:413-418). The total number of discolored internodes greater than 75% (antgr75) was recorded in the first five internodes (Figure 20). This provided a disease score ranging from 0 to 5, with zero indicating no internode more than 75% discolored and 5 indicating complete discoloration of the first five internals. The two central plots that were harvested via combination of physiological maturity and grain yield in kg/ha were determined.

Summary results over all locations are shown in Figure 12 for disease severity and Figure 13 for yield. Data show that hybrids containing Rcgl (DE811ASR (BC5) / B73Ht and DE811ASR (BC5) / Mol7Ht) have disease progression, much less than hybrids without Rcgl (DE811/B73Ht and DE811/Mol7Ht). The high score for disease progression in the susceptible hybrids (without Rcgl) shows the successful infection of the Cg experiment. Furthermore, the data show that, when infected with Cg, hybrids containing the Rcgl locus have a higher yield than the hybrid without Rcgl. Differences from individual pairwise comparisons are significant at P < 0.05.

These results clearly demonstrate that, using the methods of the embodiments, a hybrid can be created that produces more kilograms of grain per hectare when infected with Cg.

Example 14

Pure Rcgl locus conversions and hybrids derived from DE811ASR (BC5) or MP305 are resistant to stem rot induced by the fungus Colletotrichum graminicola

In order to demonstrate that commercial maize lines can be resistant to Cg-induced stalk rot, MP305 and DE811ASR (BC5) were crossed with PH87P, PH5W4 and PH705. The resulting progeny were backcrossed to the same three lines (i.e., the lines were used as recurrent parents in a backcrossing program) three more times, each time selecting for the presence of the Rcgl locus using molecular markers, as described in Example 5 As controls, selected backcross lines that lost the Rcgl locus were also collected from the same backcross program. After three backcrosses were completed, several versions were selected and selfed to obtain BC3S1 families. Individual BC3S1 plants were genotyped and homozygous positive and homozygous negative plants for Rcgl were self-pollinated to obtain BC3S2 families, which were then phenotyped. BC3S2 versions containing Rcgl and, as controls, selected versions lacking the gene were planted in single-line plots, containing approximately 25 plants per row. The experiment was planted at five different locations in five different states in the United States, designated Sites 1, 2, 3, 4 and 5. Approximately two weeks after flowering, the plants were inoculated with Cg at the base of the stem. Four to five weeks later, the stems were divided and disease progression assessed visually by estimating the amount of disease in the stem. A visual score was assigned to each stem based on the degree of infection of each internode for the inoculated internode and the four internal ones above the inoculated internode. A low score therefore indicates resistance to the disease. The results compiled for all strains and sites are summarized in Figure 14. Representative photos of the two strains are shown in Figures 15 and 16. The data shows that across all sites, each of the elite inbred strains became more resistant to the disease with the presence of the Rcgl locus.

Corn seed sold to farmers is "hybrid," meaning it is most commonly the result of a cross of two parental strains, referred to as a simple hybrid. Many years of breeding and production experience have shown that the use of simple hybrids results in higher yields. This is important for commercial applications that gene function

Rcgl in hybrid plants (that field in the farmer's production), even when it is present in only one of the two parents used to make a single crossed hybrid seed. One of the inbred lines into which the Rcgl line had been crossed, PH705, was thus used to create hybrid seeds from the cross with PH4CV, an elite inbred that does not carry the Rcgl locus. The resulting hybrid seeds were used in experiments identical to those described for the inbred lines as discussed above and scored the same at all five sites. Data is summarized for all sites in Figure 17, which also shows the performance of pure PH705, and representative images indicated in Figures 18 and 19. As can be seen, a clear difference in disease progression was observed at all sites in the hybrid PH705 x PH5W4 and in four of the five positions for PH705 x PH87P. At the fifth site, environmental conditions were too stressful for plant growth, resulting in plants that were in poor condition. Under these conditions, plant disease resistance measurements are often unreliable.

The results with the two Rcgl-containing lines and hybrid combinations clearly demonstrate that the methods of the embodiments can create commercially useful lines that are resistant to Cg-induced stalk rot.

Example 15

Markers within the Rcgl coding sequence, local markers and projects within the Rcgl locus, and haplotypes for the flanking chromosome region

Three levels of local markers can be used as a result of fine mapping and cloning of the Rcgl and Rcglb genes: markers designed within the Rcgl or Rcglb coding sequences, markers designed within the non-collinear region that identifies the Rcgl locus (but outside the Rcgl and Rcglb coding sequences), and markers designed within the collinear flanking region.

After identification and fine mapping of the Rcgl gene, hybridization markers were designed that will work on SNP platforms. Because the Rcgl gene occurs in a non-collinear region of the maize genome, the hybridization marker will be present in lines that contain the Rcgl gene and absent in lines that do not comprise the Rcgl gene. These markers identify polynucleotide sequences specific to the Rcgl coding sequence listed in SEQ ID NO: 1. As noted in Table 7, there are maize lines with other variants of the Rcgl coding sequence further in SEQ ID NO: 1, and these markers were also designed to also identify these Rcgl coding sequence variants.

To achieve this, a consensus map of Rcgl variant coding sequence from different sources was created, as shown in Table 7. This consensus map aligned 4209 bases of the Rcgl coding sequence isolated from MP305 with 3451 bases of PHBTB and 3457 bases of PH26T. The Rcgl gene in both PHBTB and PH2 6T shows resistance to anthracnose. Next, segments of the Rcgl coding sequence were compared using BLAST ® against diverse databases including NT (NCBI Public DNA) and the highest homology was aligned with the Rcgl consensus sequence to determine segments that share high homology and had common segments with other resistance genes in the NBS-LRR family. Regions unique to the Rcgl coding sequence and common among different sources of Rcgl were selected for marker design. Specifically, since primers FLP111F and FLP111R produced a single fragment that actually diagnosed the presence of Rcgl from different sources, the regions where FLP111F and FLP111R hybridized were therefore targeted for the development of a SNP marker design.

An INVADER™ detection system marker was designed using a 1413bp segment of the consensus sequence that contained both primer sites, with the primer regions being targeted for probe and oligo hybridization by INVADER™'. Primers were designed around each marker site. test to give an amplicon of size below 150 bp. This marker indicated the presence of the Rcgl coding sequence with fluorescence, due to hybridization, with the absence of the Rcgl coding sequence resulting in no fluorescence. A control fluorescence signal can also be generated by designing a marker that crosses with a second, highly conserved maize gene, such that the presence of the Rcgl coding sequence results in the fluorescence of two dyes (Rcgl and the conserved gene ) and the absence of Rcgl results in fluorescence due only to the conserved gene. This "fluorescence" control can be used to reduce laboratory errors by distinguishing between situations in which Rcgl is in fact absent and the situation in which a false negative has occurred because of a failed reaction. Such indicators are not limited to a specific marker detection platform. TaqMan® markers were also designed for the same location (primer pairs FLP111F and FLP111R), which were used as for the INVADER™ detection system markers. The markers are presented in Table 15 and Figures 23 and 24.

Marker designs C00060-01-A and C00060-02-A were tested on a wide variety of sources and were highly successful in identifying plants that contained the Rcgl locus and the Rcgl gene, regardless of the origin of the Rcgl locus or gene. Rcgl. These markers were also used against a set of controls from nearly 100 different known strains that do not have the gene, and no fluorescence was detected in the control set. Plants in which one or both drawn markers C00060-01-A and C00060-02-A confirmed to have Rcgl include those shown in Table 7.

Therefore, this example shows that, based on the teachings provided therein, markers can be constructed to identify the Rcgl coding sequence in a variety of sources. Markers within the Rcgl locus

Markers can be designed for the Rcgl locus in addition to or instead of using markers within the Rcgl or Rcglb coding sequences. The short physical distance between the Rcgl coding sequence and the non-collinear region makes it unlikely that linkage between markers within the non-collinear region but outside the Rcgl coding sequence will be lost through recombination. As with markers for the Rcgl coding sequence, a marker that shows as present or absent would be sufficient to identify the Rcgl locus.

To design markers for this region, a 64,460 bp segment of the non-collinear region including the Rcgl gene and the region north of the Rcgl gene (thus, between Rcgl and Rcglb) was initially sequenced. BACs in this sequence were divided into subclones approximately 800 nucleotides in length and sequenced. These sequences were then assembled to construct the BAC sequence and genic and repetitive regions were identified. Repetitive regions were identified in order to avoid placing markers in repetitive regions. Likewise, groups with high homology to known maize sequences were easily avoided by a simple BLAST search. Potential sequences were avoided that contained SSRs, wheel of As, Ts or Gs, or that resulted in the generation of low Cg content probes that could cause problems with the INVADER™ detection system platform. See Figure 9 (b) and Table 17.

Selected segments were then placed into INVADER CREATOR™ software, which generates oligos for an INVADER™ reaction. This produced a sense and anti-sense project of all SNPs. The drawings felt with the best scores and penalties were not selected. Although these markers have been designed, they have not yet been tested.

Primers were designed using Primer3 (Steve Rozen and Helen J. Skaletsky (2000). Primer3 is available on the World Wide Web for general users and programming biologists. See, Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Human Press, Totowa, NJ, pp 365-386. Primers were selected from outside of the INVADER™ components and preferred primers near or below 150 bp in length were selected to be most useful. for the INVADER™ detection system platform, despite using other detection platform primers, it would be optimized for use with these platforms Collinear Region Markers and Associated Haplotypes.

Closely linked markers flanking the Rcgl locus can be effectively used to select a progeny plant that has inherited the Rcgl locus from a parent that comprises the Rcgl locus. The markers described here, such as those listed in Table 16, as well as other markers genetically or physically mapped to the same chromosomal segment, can be used to select a truncated chromosomal segment that makes up the Rcgl locus. Typically, a set of these markers will be used (e.g., 2 or more, 3 or more, 4 or more, 5 or more) in the flanking region above the gene and a similar flanking set in the region below the gene. Optionally, as described above, a marker from within the Rcgl gene, the Rcglb gene and/or the Rcgl locus can also be used. Parents and their offspring are selected for these sets of markers, and markers that are polymorphic between the two parents are used for selection. The polymorphic markers most proximal to the Rcgl gene, the Rcglb gene, or the Rcgl locus are used to select for the gene or locus, and the most distal polymorphic markers are used to select against the gene or locus. In an introgression program, this allows selection for the Rcgl gene, the Rcglb gene, or Rcgl locus genotype of the most proximal polymorphic markers and selection for the recurrent parental genotype at the most distal polymorphic markers. As described in more detail in Example 5 above, this process allowed efficient selection of a truncated chromosomal segment that makes up the Rcgl locus.

The process described above requires knowledge of the parental genotypes used in the crossing. Optionally, haplotypes can be used so that the Rcgl gene, Rcglb gene or Rcgl locus can be selected for first genotyping without the specific parents used in the cross. This is a highly efficient way to select the Rcgl locus, especially in the absence of using markers on the Rcgl gene, Rcglb gene or the Rcgl locus.

All plants to be used in a breeding program, such as a gene introgression program, are evaluated with markers. The markers disclosed herein or equivalent markers on the same chromosome segment may be used. Plant haplotypes A (a series of SNPs or other linkage disequilibrium markers) are observed. The haplotype of the resistant plant around the Rcgl locus is compared with the haplotype of other plants to be used that do not include the Rcgl locus. A haplotype unique to the resistant plant around the Rcgl locus is then used for selection, and this haplotype will specifically identify the chromosome segment of the resistant plant with the Rcgl locus.

Based on an analysis of MP305 and a diverse set of several hundred corn lines, including the 50 public corn lines presented in Table 18, a unique SNP haplotype for the MP305 chromosomal segment with the Rcgl locus was identified. This SNP haplotype identifies the MP305 chromosomal segment that spans MZA3434, MZA2591 and MZA11123. See Figure 22, SEQ ID NO: 140, 141 and 142, and tables 8, 9 and 10.

First, the primer pairs described in Table 2 for these three MZA were used to identify haplotypes. The forward and reverse primer pairs MZA3434 E were used to amplify the genomic DNA of the set of maize lines. The PCR fragments were further purified by amplification with forward and reverse primer pairs MZA3434. This process was repeated for MZA2591 and MZA11123. The resulting PCR fragments were sequenced in the forward and reverse direction, and the sequences were aligned to give a consensus sequence (see the sequences set forth in SEQ ID NOs: 140, 141 and 142). SNPs and indels within these consensus sequences are shown in Tables 8, 9 and 10. These series of SNPs and indels were compared across the genotype set.

For MZA3434, haplotype 8 was a rare allele haplotype, and was the only one for MP305 and only one other maize line. This process was repeated for MZA2591 and MP305 was found to have haplotype 2 in MZA2591, which was shared by only two other maize lines. MP305 was the only maize line to have both haplotype 8 in MZA3434 and haplotype 2 in MZA2591, and therefore the combination of these two haplotypes, 8 in MZA3434 and 2 in MZA2591, identifies the MP305 chromosomal region that makes up the Rcgl locus. MP305 also had an informative haplotype in MZA11123. MP305 was found to have haplotype 7, which was shared by 66 other maize lines, but none of these maize lines had haplotype 8 in MZA3434, or haplotype 2 in MZA2591. Therefore, any combination of 2 haplotypes in MZA3434, MZA2591 or MZA11123 could be used to identify MP3 05 among these genotypes. Haplotypes can then be defined by sequencing the fragment or by designing markers for each SNP or Indel within a fragment.

Polymorphisms in haplotypes can be used to mark the haplotype. So called "Tag-SNPs", or "haplotype tags" can be very useful in plant breeding, so more information from the polymorphism itself can be determined through extrapolation to the haplotype. A haplotype can also be defined as a series of polymorphisms in sequences, and these can be termed "long-range haplotypes".

Rare polymorphisms have been observed within haplotypes that could be used as "tag haplotypes". For example, either the MZA2591.32 SNPs (c allele) or MZA2591.35 (t allele) could be used to mark haplotype 2 in MZA2591, and as haplotype 2, both were unique to MP305 and two other maize lines. The combination of SNPs MZA2591.32 (c allele) and MZA2591.35 (t allele), combined with MZA3434.17 (c allele) gave a "long-range haplotype" that could be used to distinguish MP305 from all other genotypes in study.

In addition, other markers, MZA15842, MZA11455, MZA8761 and MZA1851 also showed polymorphism with MP305. For MZA15842, only 18 of the other maize lines shared the same haplotype as MP305; for MZA11455, only 43 of the other maize lines shared the same haplotype as MP305; for MZA8761, only about half of the other maize lines shared the same haplotype as MP305, and for MZA1851, only about half of the other maize lines shared the same haplotype as MP305. Consensus sequences have been developed for these markers, and are set forth in SEQ ID NOs: 143-146. SNPs and indels within these consensus sequences are shown in Tables 11-14. Four examples of unique haplotypes using the MZA markers are: MZA11123 haplotype (7) type ( 7) MZA15842 haplotype (3) MZA16510 haplotype (4) and MZA11455 haplotype (6) MZA11123 haplotype (7) MZA15842 haplotype (3) MZA11394 haplotype (6) Various combinations within all disclosed markers, or other markers within the region, contain unique haplotypes that identify the locus Table 16: Markers contained within defined chromosomal intervals that can be used to select for Rcgl.

The public markers are taken from the IBM2 4 neighbor map, while the relative positions of the Pioneer 5 markers (prefix 'MZA') were determined by mapping to the same genetic map, and by location on the physical map. Table 18: List of Public Lineages used in Haplotype Analysis

Claims (21)

1. Isolated polynucleotide characterized by comprising a polynucleotide operatively linked to a heterologous regulatory sequence selected from the group consisting of: (a) the nucleotide sequence SEQ ID NO: 245 encoding a polypeptide capable of conferring or increasing resistance to Colletotrichum when co- expressed in a plant with the nucleotide sequence SEQ ID NO: 2 encoding SEQ ID NO: 3, where the polypeptide has the amino acid sequence SEQ ID NO: 246; (b) a complement of the nucleotide sequence (a), where the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary. 2. Vector characterized by comprising the polynucleotide of claim 1. 3. Process for transforming a host cell, characterized in that said process comprises: a) transforming the host cell, with at least one polynucleotide selected from the group consisting of: i) SEQ ID NO: 245; and ii) SEQ ID NOs: 2 and 245; b) cultivating said transformed host cell under host cell growth conditions, wherein said transformed host cell exhibits resistance to Colletotrichum. 4. Process according to claim 3, characterized in that the host cell is a plant cell. 5. Process according to claim 4, characterized in that the plant cell is corn. 6. Process for producing a plant characterized in that said process comprises: a) transforming a plant cell with the polynucleotide of claim 1; b) cultivating said transformed plant cell under plant cell growth conditions; and c) regenerating a plant from said transformed plant cell; in which said plant presents resistance to Colletotrichum. 7. Process according to claim 6, characterized in that said plant is corn. 8. Process for conferring or improving resistance to Colletotrichum, characterized in that it comprises transforming a plant with the polynucleotide of claim 1, thereby conferring or improving resistance to Colletotrichum. 9. A process for altering the level of expression of proteins capable of conferring resistance to Colletotrichum in a plant cell, characterized by comprising: (a) transforming a plant cell with the polynucleotide according to claim 1, and (b) culturing the transformed plant cells under conditions that are suitable, wherein said conditions result in the production of altered levels of proteins capable of conferring resistance to Colletotrichum in the transformed plant cell. 10. Process for altering the level of expression of proteins capable of conferring resistance to rot in the stem of a plant cell, characterized by comprising: (a) transforming a plant cell with the polynucleotide of claim 1, and (b) cultivating the plant cells transformed under conditions that are suitable, wherein said conditions result in the production of altered levels of proteins capable of conferring resistance to stem rot of transformed plant cells. 11. Process for altering the level of expression of proteins capable of conferring resistance to Colletotrichum in a plant characterized by comprising: (a) transforming a plant cell with the polynucleotide of claim 1, and (b) regenerating a transformed plant from the cell transformed plant and (c) cultivating the transformed plant under conditions that are suitable, wherein said conditions result in the production of altered levels of proteins capable of conferring resistance to Colletotrichum in transformed plants. 12. Process for altering the level of expression of proteins capable of conferring resistance to stem rot in a plant, characterized by comprising: (a) transforming a plant cell with the polynucleotide of claim 1, and (b) regenerating the transformed plant to from the transformed plant cell, and (c) cultivating the transformed plant under conditions that are suitable, wherein said conditions result in the production of altered levels of proteins capable of conferring resistance to stem rot in the transformed plant. 13. Process of identifying a corn plant that presents greater resistance to infection by Colletotrichum, the process comprising detecting in the corn plant the presence or absence of at least one marker at the Rcg1 locus, characterized in that said at least one marker is on or within a polynucleotide sequence selected from the group consisting of SEQ ID NOS: 255, 256, 257, 258 and 266 and selecting the corn plant in which said at least one marker is present. 14. Process according to claim 13, characterized by further comprising detecting the presence or absence of a second marker in or within a sequence selected from the group consisting of: a. SEQ ID NO: 137; B. SEQ ID NO: 263; w. SEQ ID NO: 264; d. SEQ ID NO: 265; and is. SEQ ID NO: 266. 15. Process according to claim 13, characterized in that at least one marker is at or within SEQ ID NO: 245 (the Rcg1b coding sequence). 16. The method of claim 15, further comprising a second marker in or within SEQ ID NO:245 (the coding sequence Rcg1). 17. Process according to claim 15, characterized in that the Rcg1b coding sequence (SEQ ID NO: 245) comprises a nucleotide sequence that encodes a polypeptide, where the polypeptide has the amino acid sequence SEQ ID NO: 246. 18. The method of claim 16, wherein at least one marker that is in or within the polynucleotide sequence is in or within a polynucleotide sequence selected from the group consisting of SEQ ID NOS: 255, 256, 257, 258 and 266. 19. Process according to claim 18, characterized by further comprising a second marker on or within the polynucleotide set forth in SEQ ID NO: 1. 20. Process according to claim 13, characterized in that at least one marker detects a polynucleotide comprising a nucleic acid sequence as described in SEQ ID NO: 245 or a complement thereof. 21. The method of claim 20, further comprising a second marker that detects a polynucleotide comprising a nucleic acid sequence as described in SEQ ID NO: 245 or a complement thereof.