CN113121664A - Method for identifying, selecting and generating disease resistant crops - Google Patents

Method for identifying, selecting and generating disease resistant crops Download PDF

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CN113121664A
CN113121664A CN202011200759.6A CN202011200759A CN113121664A CN 113121664 A CN113121664 A CN 113121664A CN 202011200759 A CN202011200759 A CN 202011200759A CN 113121664 A CN113121664 A CN 113121664A
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赖志兵
王宏泽
候甲宝
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Huazhong Agricultural University
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Abstract

The field relates to plant breeding and methods of identifying, selecting and producing disease resistant crops. Methods of identifying novel genes encoding proteins that provide plant disease resistance and uses thereof are provided. These disease resistance genes can be used for the generation of resistant plants by breeding, transgene modification or genome editing.

Description

Method for identifying, selecting and generating disease resistant crops
Reference to electronically submitted sequence Listing
A sequence listing filed in computer-readable form with this specification with the file name "RTS 22658A _ seqlist. txt", created at 10, 15/2020, and having a size of 114 kilobytes. The sequence listing is part of this specification and is incorporated herein by reference in its entirety.
Technical Field
The field relates to plant breeding and methods of identifying and selecting plants with disease resistance. Methods of identifying novel genes encoding proteins that provide plant disease resistance and uses thereof are provided. These disease resistance genes can be used for the generation of resistant plants by breeding, transgene modification or genome editing.
Background
Southern rust (SCR) of maize is a fungal disease caused by Puccinia polysorum Underw, a major disease in tropical regions as well as in the united states and south of china. If the SCR reaches the critical point in the temperate zone (e.g., the midwest of the united states) during growth and if conditions favor the development of rust, the disease intensity can quickly reach epidemic levels, resulting in severe yield loss. Temperate corn germplasm is typically susceptible to SCR. The identification and use of resistance lines and QTLs in breeding programs to develop varieties resistant to SCR represents an economically efficient way to control SCR. Alternatively, varieties carrying genes responsible for SCR resistance can be developed by transgenic or genome editing techniques. Identifying resistance QTLs and genes will accelerate the development of resistance of the product to SCR. Resistant lines (e.g., Brewbaker, J.L., et al, "General resistance to Southern rust (maize multislice.) ] Crop science 51, stage 4 (2011): 1393) 1409) or QTLs (e.g., Jines, M.P., et al," Mapping resistance to Southern rust in a tropical by maize recombinant inbred population "] the resistance location of the tropical Southern rust by the temperate maize recombinant inbred population ] the genetic and Applied Genetics [ Theoretical and Applied Genetics ]114, stage 4 (2007) 659-J.L., Zhang. Y., Zhang et al, [ Southern rust [ gene of located of maize ] the Southern rust [ Southern rust of Zephyte genetic of [ maize of 5 ] Molecular Mapping of Zephyte in Southern rust of Zephys ]83, stage 3 (2010): zhou CJ, et al (2007) Characterization and fine mapping of RppQ, a resistance gene to southern corn rust [ Characterization and fine localization of RppQ, resistance genes of maize to southern corn rust ] Mol Genomics [ molecular genetics and Genomics ] 278: 723-728 Holland, J.B., et al, "heredity of resistance of maize populations with tropical maize to southern maize rust" Thetical and Applied Genetics [ theories and Applied Genetics ]96, 2 (1998): 232-241.). However, the causative genes responsible for SCR resistance have not been identified and characterized. There is a continuing need for disease resistant plants and methods of finding disease resistance genes.
Disclosure of Invention
Provided herein are compositions and methods useful in identifying and selecting plant disease resistance genes, or "R genes". The compositions and methods are useful for selecting disease resistant plants, producing transgenic resistant plants, and/or producing plants with resistant genome editing. Also provided herein are plants having newly conferred or enhanced resistance to various plant diseases as compared to control plants. In some embodiments, the compositions and methods can be used to select disease resistant corn plants, including corn southern rust (SCR) disease resistant plants, to produce transgenic disease resistant plants, and/or to produce plants with disease resistant genome editing.
The disease resistant plant can be crossed with a second plant to obtain a progeny plant having the resistance gene allele. The disease resistance may be newly conferred or enhanced relative to a control plant not having the favorable allele. The R gene allele may be further refined to a chromosomal interval defined by and including the defined marker. In some embodiments, methods for identifying and/or selecting plants that are disease resistant are presented. In these methods, the DNA of the plant is analyzed for the presence of a resistance gene allele associated with disease resistance on chromosome 7, wherein the resistance gene allele comprises a nucleotide sequence that is identical to the nucleotide sequence of SEQ ID NO: 1-10, or 13-16, sequences that are at least 95% identical; and identifying and/or selecting the plant as disease resistant if the resistance gene allele is detected. In some embodiments, the method for identifying and/or selecting plants that are resistant to disease comprises detecting or selecting a plant comprising SEQ ID NO: 4-6. The disease resistance may be newly conferred or enhanced relative to a control plant not having the favorable allele. In another embodiment, the disease resistance region comprises a gene encoding a ZmMM1 polypeptide that confers or enhances disease resistance ("ZmMM 1" gene). In some embodiments, the ZmMM1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1-3, or a pharmaceutically acceptable salt thereof.
In another embodiment, a method of identifying and/or selecting plants having disease resistance is provided, wherein one or more genes that are identical to SEQ ID NO: 1-10 or 13-16, and selecting for plants having one or more marker alleles. The one or more marker alleles may be linked at 10cM, 9cM, 8cM, 7cM, 6cM, 5cM, 4cM, 3cM, 2cM, 1cM, 0.9cM, 0.8cM, 0.7cM, 0.6cM, 0.5cM, 0.4cM, 0.3cM, 0.2cM, or 0.1cM or less on a single meiosis based genetic map. The selected plant can be crossed with a second plant to obtain a progeny plant having a nucleotide sequence identical to SEQ ID NO: 1-10 or 13-16.
In another embodiment, a method of introgressing an allele of a gene associated with disease resistance is presented herein. In these methods, a population of plants is screened with one or more markers to determine if any of the plants has a genetic allele associated with disease resistance, and at least one plant having a genetic allele associated with disease resistance is selected from the population. The allele comprises a nucleotide sequence identical to SEQ ID NO: 1-10 or 13-16 sequences having at least 95% identity.
In some embodiments, introgression of the disease resistance gene from a resistant strain into a susceptible strain can be accomplished by marker assisted trait introgression, transgenesis, or genome editing methods.
Embodiments include isolated polynucleotides comprising a nucleotide sequence encoding a ZmMM1 polypeptide capable of conferring disease resistance, wherein the ZmMM1 polypeptide has an amino acid sequence identical to SEQ ID NO: 1-3, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity when compared. In another embodiment, the isolated polynucleotide comprises a nucleotide sequence encoding a ZmMM1 polypeptide capable of conferring resistance, wherein the ZmMM1 polypeptide has an amino acid sequence identical to SEQ ID NO: 1-3, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% identical when compared.
Additional embodiments of the disclosure include polynucleotides comprising the disclosure, e.g., SEQ ID NO: 4-10, or a recombinant DNA construct comprising a polynucleotide disclosed herein operably linked to at least one regulatory sequence. Plant cells and plants each comprising the recombinant DNA constructs of the embodiments disclosed herein, and seeds comprising the recombinant DNA constructs, are also presented.
In some embodiments, the compositions and methods relate to modified plants with increased resistance to disease, wherein the allele that causes increased disease resistance comprises a nucleotide sequence encoding a ZmMM1 resistance gene, wherein the ZmMM1 resistance gene is homologous to the nucleotide sequence of SEQ ID NO: 4-10 have at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity. In some embodiments, down-regulation in maize of ZmMT1(SEQ ID NOs: 20 and 21), ZmMT2(SEQ ID NO: 23), ZmMT3, or ZmMT4(SEQ ID NO: 25) provides enhanced resistance. Downregulation can be induced by editing or transgenic means (including RNAi knockouts).
Methods embodied by the present disclosure relate to methods for transforming a host cell, including a plant cell, comprising transforming a host cell with a polynucleotide of one embodiment of the present disclosure; for making plants comprising transforming a plant cell with a recombinant DNA construct of one embodiment of the disclosure and regenerating a plant from the transformed plant cell, and conferring or enhancing disease resistance, the method comprises transforming a plant with the recombinant DNA construct disclosed herein.
Also presented are methods of altering the level of expression of a protein capable of conferring disease resistance in a plant or plant cell, comprising (a) transforming a plant cell with a recombinant DNA construct disclosed herein, and (b) culturing the transformed plant cell under conditions suitable for expression of the recombinant DNA construct, wherein expression of the recombinant DNA construct results in the production of altered levels of the protein capable of conferring disease resistance in a transformed host.
Also provided are plants identified and/or selected using any of the above methods.
Brief description of the drawings
FIG. 1 shows the 5kb interval with fine localization of the flanking markers M2(SEQ ID NOS: 27 and 28) and M3(SEQ ID NOS: 11 and 12) on chromosome 7 and 20 SNPs and 7 indels between C117 and Mo17 within the 1kb qLMchr7 region.
DESCRIPTION OF THE SEQUENCES
Figure BDA0002752275220000051
Figure BDA0002752275220000061
Detailed Description
As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "a protein" includes reference to one or more proteins and equivalents thereof, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs unless explicitly stated otherwise.
The NBS-LRR ("NLR") group of R genes is the largest class of R genes discovered to date. In Arabidopsis thaliana (Arabidopsis thaliana), more than 150 NLR genes are expected to be present in the genome (Meyers et al, (2003), Plant Cell [ Plant cells ], 15: 809-834; Monosi et al, (2004), theractic and Applied Genetics [ theory and Applied Genetics ], 109: 1434-1447), whereas in Oryza sativa, approximately 500 NLR genes have been predicted (Monosi, (2004) supra). The NBS-LRR class of R genes consists of two subclasses. The 1-type NLR gene contains a TIR-Toll/interleukin-1-like structural domain at the N' end; they have been found to date only in dicotyledonous plants (Meyers, (2003) supra; Monosi, (2004) supra). The second class of NBS-LRRs contains a coiled-coil domain or (nt) domain at their N-terminus (Bai et al (2002) Genome Research [ 12: 1871-. Class 2 NBS-LRRs are found in both dicot and monocot species. (Bai, (2002) supra; Meyers, (2003) supra; Monosi, (2004) supra; Pan, (2000) supra).
The NBS domain of this gene appears to play a role in signaling of plant defense mechanisms (van der Biezen et al, (1998), Current Biology [ Current Biology ]: CB, 8: R226-R227). The LRR region appears to be the region of interaction with the pathogen AVR product (Michelmore et al, (1998), Genome Res. [ Genome research ], 8: 1113-1130; Meyers, (2003) supra). The LRR region is subject to greater selection pressure for diversification than the NB-ARC (NBS) domain (Michelmore, (1998) supra; Meyers, (2003) supra; Palomino et al, (2002), Genome Research [ Genome Research ], 12: 1305-. LRR domains may also be found in other contexts; these 20-29 residue motifs are arranged in tandem in many proteins that have multiple functions, such as hormone-receptor interactions, enzyme inhibition, cell adhesion and cell trafficking. Many recent studies have shown that LRR proteins are involved in early development, neural development, cellular polarization, regulation of gene expression, and apoptosis signaling in mammals.
An allele is "associated with" a trait when it is part of or linked to a DNA sequence or allele that affects the expression of the trait. The presence of the allele is an indicator of how the trait will be expressed.
As used herein, "disease resistance" or "resistance to disease" refers to a plant that exhibits increased resistance to disease as compared to a control plant. Disease resistance may manifest as fewer and/or smaller lesions, increased plant health, increased yield, increased root mass, increased plant vigor, less or no discoloration, increased growth, reduced necrotic area, or reduced wilting. In some embodiments, the allele can be shown to be resistant to one or more diseases.
Diseases affecting maize plants include, but are not limited to, bacterial leaf blight and stem rot (bacterial leaf blight and talk rot); bacterial leaf spot; bacterial streak (bacterial strip); red spot disease (crocolate spot) of fava bean; bacterial blight and blight (goss's bacterial wilt and blight); chorionic alternaria leaf spot (holcus spot); leaf sheath purpura (purpura leaf sheath); seed rot-seedling blight (seed rot-seed blast); bacterial wilt disease (bacterial witt); maize dwarf disease (corn stunt); anthracnose leaf blight (anthracnose leaf light); anthracnose stem rot (anthracnose stalk rot); aspergillus ear and kernel rot (aspergillus ear and kernel rot); banded leaf and sheath spot (bandled leaf and sheath spot); black bundle disease (black bundle disease); black kernel rot (black kernel rot); white border disease (Borde blanco); brown spot; black spot (black spot); stem rot (stalk rot); cephalosporium granulosis (cephalosporium kernel rot); charcoal rot (charcola rot); cornucopia ear rot (corticium ear rot); curvularia leaf spot; subacute septate leaf spot (dymelalla leaf spot); ear rot and stem rot of diplodia (diplodia ear rot and talk rot); diplodia ear rot (dipnodia ear rot); seed rot; maize seedling blight (corn seed blast light); diplodia leaf spot or leaf streak (dipodia leaf spot or leaf streak); downy milews; downy mildew of brown streak; top locoweed downy mildew (crazy top downy milew); green ear downy mildew (green ear downy milew); gramineous downy mildew (graminicola downy milew); java downy milew; philippine downy milew; sorghum downy mildewe (sorghum); downy mildew of sweet grass; sugarcane downy mildew (sugarcane downy milew); dry ear rot (dry ear rot); ergot (ergot); horse's tooth disease; corn eyespot (corn eyespot); fusarium ear and stalk rot (fusarium ear and talk rot); fusarium wilt disease (fusarium blight); seedling root rot (seedling root rot); gibberella ear and stem rot (gibberella ear and talk rot); gray ear rot (gray ear rot); gray leaf spot; cercospora leaf spot (cercospora leaf spot); helminthosporium root rot; ramaria monascus (hormodendrum ear rot); mycosporic rot (cladosporium rot); leaf spot of Hypericum perforatum (hylothyridium leaf spot); late blight (late salt); northern leaf blight (northern leaf blast); white blast (white blast); crown stem rot (crown stalk rot); corn streak (corn streak); northern leaf spot; helminthosporium ear rot (helminthosporium ear rot); penicillium ear rot (penicillium ear rot); corn blue eye disease (corn blue eye); downy mildew (blue mold); ascophyllum melanosporum stem rot and root rot (phaeocytotropha stalk rot and root rot); mycosphaerella globiformis leaf spot (phaeosphaeria leaf spot); ascomycetous ear rot (physiospora ear rot); botrytis cinerea ear rot (botryosphaeria ear rot); ascochyta stem rot and root rot (Pyrenochaeta stalk rot and root rot); pythium root rot; pythium stem rot (pythium stalk rot); red kernel disease (red kernel disease); rhizoctonia ear rot (rhizoctonia ear rot); sclerotinia rot (sclerotirotial rot); rhizoctonia root rot and stem rot (rhizoctonia root rot and stalk rot); leaf spot of rostratum (rostratum leaf spot); common type corn rust (common rust); southern corn rust (southern corn rust); tropical corn rust (tropical corn rust); sclerotium rot (sclerotiotium ear rot); southern blight (southern bright); leaf spot of Sporochaeta (selenophoma leaf spot); sheath rot (sheath rot); shell rot (shuck rot); silage mold (silage mold); smut (common smut); false smut (false smut); head smut; southern corn leaf blight and stem rot disease (southern corn leaf blast and talk rot); southern leaf spot; black tumor (tar spot); trichoderma ear rot and root rot (trichoderma ear rot and root rot); white ear rot, root and stem rot; yellow leaf blight (yellow leaf blight); leaf spot of mulberry (zonate leaf spot); american wheat streak mosaic (wheat streak mosaic); barley stripe mosaic (barley stripe mosaic); wheat yellow dwarf (barley yellow dwarf); brome mosaic (broome mosaic); cereal chlorosis mottle (cereal chlorotic mottle); fatal necrosis (maize fatal necrosis disease); cucumber mosaic (cucumber mosaic); johnsongrass mosaic virus (johnsongsasmosaic); maize bushy stunt; maize chlorosis dwarf (maize chlorosis dwarf); maize chlorosis mottle (maize chlorotic mottle); maize dwarf mosaic (maize dwarfmosaic); maize blight spot (maize leaffleck); corn clear ring spot (maize pellucid ringspot); maize rafado fina (maize rayado); zea mays red leaf and red streak (red leaf and red stripe); maize red streak (maize red stripe); maize ring spot disease (maize ring mottle); maize rough dwarf (maize rough dwarf); maize sterile stunt (maize stereo stunt); maize streak disease (maize streak); maize streak (maize streak); maize tassel aberration (maize tassel aberration); maize vein protrusion (maize vein enation); maize murine ear disease (maize wallaby ear); maize white leaf disease (maize white leaf); maize white line mosaic (maize white line mosaic); millet red leaf disease (millet red leaf); and northern cereal mosaic (northern cereal mosaic).
Diseases affecting plants include, but are not limited to, bacterial wilt disease (bacterial light); bacterial leaf streak (bacterial leaf streak); basal rot (foot rot); grain rot disease (grain rot); brown sheath disease (sheath brown rot); blast disease (blast); brown spot; crown sheath rot (crown sheath rot); downy mildew (downy mildew); ocular spot disease (eyespot); false smut (false smut); kernel smut (kernel smut); leaf smut (leaf smut); leaf scald (leaf scald); narrow brown leaf spot; root rot; seedling blight (seedling blast); sheath blight (sheath bright); sheath rot (sheath rot); sheath spot disease (sheath spot); alternaria leaf spot (alternaria leaf spot); and stem rot (stem rot).
Diseases affecting soybean plants include, but are not limited to, alternaria leaf spot; anthracnose (anthracnose); black leaf blight (black leaf bright); black root rot (black root rot); brown spot; brown stem rot (brown stem rot); charcoal rot (charcola rot); choanephora leaf blight (choranephora leaf bright); downy mildew (downy mildew); helmholra blight (drechslera blight); frog-eye leaf spot (frog eye leaf spot); leptospermum leaf spot (leptosporaneous leaf spot); mycophylaxis root rot (mycophylaxis root rot); new red husk stem rot (neocomospora stem rot); phomopsis seed rot (phomopsis seed decay); phytophthora root and stem rot (phytophthora root and stem rot); leaf spot of phyllosticta leaf; rhizomatosis root rot (phymatotrichum root rot); black spot disease (pod and stem light); powdery mildew (powdery mildew); purpura (purple seed stain); acanthosporium leaf spot (pyrenochaeta leaf spot); pythium rot; red crown rot (red crown rot); sclerotinia sparsa leaf spot (dactuliophora leaf spot); rhizoctonia aeroginosis (rhizoctonia aerial blast); rhizoctonia root and stem rot (rhizoctonia root and stem rot); rust (rust); scab disease (scab); sclerotinia stem rot (sclerotirotinia stem rot); sclerotinia sclerotiorum (sclerotiotium blight); stem ulcers (stem canker); stemphylium leaf blight (stemphyllium leaf bright); sudden death syndrome (sudden death syndrome); wheel spot (target spot); yeast spot (yeast spot); lanse nematodes (lane nematodes); nematodes (replacement nematodes); nematodes (pin nematodes); reniform nematodes (reniform nematodes); nematode (ring nematode); root-knot nematodes (root-knot nematodes); sheath nematodes (sheath nematode); cyst nematodes (cyst nematodes); nematode (nematode) gyrus; nematoda (sting nematoda); root-knot nematode (stubbby root nematode); dwarf nematodes (stunt nematode); alfalfa mosaic (alfalfalfa mosaic); bean pod mottle disease (bean pod mottle); bean yellow mosaic (bean yellow mosaic); brazilian shoot blight (brazilian bud light); chlorotic mottle; yellow mosaic (yellow mosaic); peanut mottle (peanout mottle); arachis hypogaea streak (peanout strip); peanut dwarfing (peanout stunt); chlorotic mottle; wrinkled leaf disease (crinkle leaf); dwarf (dwarf); severe dwarfing disease (severe stunt); and tobacco ring spot or bud blight (tobaco ringspot or bud bright).
Diseases affecting canola plants include, but are not limited to, bacterial black rot (bacterial black rot); bacterial leaf spot; bacterial pod rot (bacterial pod); bacterial soft rot (bacterial soft rot); scab disease (scab); crown tumor disease (crown gall); alternaria black spot (alternaria black spot); anthracnose (anthracnose); black leg disease (black leg); black mold rot (black mold rot); black rot (black root); brown girdling root rot (brown girdling root); cercospora leaf spot (cercospora leaf spot); root tumor (clubroot); downy mildew (downy mildew); fusarium wilt (fusarium wilt); gray mold (gray mold); silk rot (head rot); leaf spot; superficial leaf spot (light leaf spot); pod rot (pod rot); powdery mildew (powdery mildew); ring spot disease (ring spot); root rot; sclerotinia stem rot (sclerotirotinia stem rot); seed rot-off (seed rot); root melanoma (root toll smut); southern blight (southern bright); verticillium wilt (verticillium wilt); bacterial leaf blight (white light); white leaf spot; blight (staghead); yellow blight (yellows); crinkle virus (crinkle virus); mosaic virus (mosaic virus); flaviviruses (yellows viruses);
diseases affecting sunflower plants include, but are not limited to, apical chlorosis (apical chlorosis); bacterial leaf spot; bacterial wilt disease (bacterial witt); crown tumor disease (crown gall); erwinia stem rot and head rot; alternaria leaf blight, stalk spot and head rot; botrytis head rot; charcoal rot (charcola rot); downy mildew (downy mildew); fusarium stalk rot (fusarium stalk rot); fusarium wilt (fusarium wilt); leaf and stem rot of Myrothecium verrucaria (myrothecium leaf and stem spot); yellow blight of bottle mold (Phialophora yellows); phoma black stem disease (phoma black stem); phomopsis brown stem canker; rhizomatosis root rot (phymatotrichum root rot); phytophthora stem rot (phytophthora stem rot); powdery mildew (powdery mildew); pythium seed blight and root rot; rhizoctonia seed blight (rhizoctonia seed blight); rhizopus head rot (rhizopus head rot); sunflower rust (sunflower rust); sclerotium basal stem and root rot (sclerotirotium basal talk and root rot); septoria leaf spot (septoria leaf spot); verticillium wilt (verticillium wilt); white rust (white rust); stripe rust (yellow rust); a short sword shape; needle-shaped; (ii) a lesion; kidney shape; root tumor diseases; and chlorotic mottle;
diseases affecting sorghum plants include, but are not limited to, bacterial leaf spot; bacterial leaf streak (bacterial leaf streak); bacterial leaf streak (bacterial leaf strip); acremonium wilt (acremonium wilt); anthracnose (anthracnose); charcoal rot (charcola rot); top locoweed downy mildew (crazy top downy milew); damping-off seed rot; ergot (ergot); fusarium rhizoctonia, root and stem rot (root and stem rot); cereal storage mold (grain storage mold); gray leaf spot; posterior leaf spot (late leaf spot); leaf blight (leaflight); milo disease (milo disease); oval leaf spot (oval leaf spot); sugarcane top rot (pokkah boeng); pythium root rot; rough leaf spot; rust (rust); seedling blight and seed rot (pythium seed blight and root rot); covered kernel smut (smut); head (smut); loose kernel smut (smut); black streak (scotia strip); downy mildew (downy mildew); black tumor (tar spot); target leaf spot; and leaf spot and sheath blight (zonate leaf spot and sheath light).
A plant with disease resistance may have 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 or 100% increased resistance compared to a control plant. In some embodiments, the plant may have 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 or 100% increased plant health in the presence of the disease as compared to a control plant.
As used herein, the term "chromosomal interval" refers to a continuous linear span of genomic DNA that is present on a single chromosome of a plant. Genetic elements or genes located on a single chromosomal interval are physically linked. The size of the chromosomal interval is not particularly limited. In some aspects, genetic elements located within a single chromosomal interval are genetically linked, typically having a genetic recombination distance of, for example, less than or equal to 20cM, or alternatively, less than or equal to 10 cM. That is, two genetic elements within a single chromosomal interval recombine at a frequency of less than or equal to 20% or 10%.
In the present application, the phrase "closely linked" means that recombination between two linked loci occurs at a frequency equal to or less than about 10% (i.e., not more than 10cM apart on the genetic map). In other words, the closely linked loci have at least 90% chance of co-segregation. Marker loci are particularly useful for the subject matter of the present disclosure when they exhibit a significant probability of co-segregation (linkage) with a desired trait (e.g., resistance to southern corn rust). Closely linked loci (e.g., a marker locus and a second locus) can exhibit an interlocus recombination frequency of 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, still more preferably about 4% or less, yet more preferably about 3% or less, and still more preferably about 2% or less. In highly preferred embodiments, the relevant loci exhibit a recombination frequency of about 1% or less, for example about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or less. Two loci that are located on the same chromosome and have a distance such that recombination between the two loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25% or less) are also considered "adjacent" to each other. In some cases, two different markers may have the same genetic map coordinates. In this case, the two markers are so close to each other that recombination between the two occurs at such a low frequency that it is undetectable.
The term "crossed" or "cross" refers to a sexual cross, and involves the fusion of two haploid gametes by pollination to produce a diploid progeny (e.g., a cell, seed, or plant). The term encompasses both pollination and selfing (or self-pollination, e.g., when pollen and ovule are from the same plant) of one plant by another.
A "elite line" is any line produced by breeding for superior agronomic performance.
"exotic varieties", "tropical lines" or "exotic germplasm" are varieties derived from plants that do not belong to available elite lines or germplasm varieties. In the case of a cross between two plants or germplasm varieties, the progeny of the foreign germplasm is not closely related to the elite germplasm with which it crosses. Most commonly, the foreign germplasm is not derived from any known elite line, but is selected for the introduction of new genetic elements (typically new alleles) into a breeding program.
An "advantageous allele" is an allele (marker, QTL, gene, etc.) of a particular locus that confers or contributes to an agronomically desirable phenotype (e.g., disease resistance) and allows for the identification of plants having that agronomically desirable phenotype. A favorable allele of a marker is a marker allele that segregates from the favorable phenotype.
A "genetic marker" is a nucleic acid that is polymorphic in a population, and alleles of the genetic marker can be detected and distinguished by one or more analytical methods (e.g., RFLP, AFLP, isozymes, SNPs, SSRs, etc.). The term also refers to a nucleic acid sequence that is complementary to a genomic sequence (e.g., a nucleic acid) used as a probe. Markers corresponding to genetic polymorphisms between members of the population can be detected by methods recognized in the art. These methods include, for example, PCR-based sequence-specific amplification methods, restriction fragment length polymorphism detection (RFLP), isozyme marker detection, polynucleotide polymorphism detection by allele-specific hybridization (ASH), amplified variable sequence detection of plant genomes, autonomous sequence replication detection, simple repeat sequence detection (SSR), single nucleotide polymorphism detection (SNP), or amplified fragment length polymorphism detection (AFLP). Known and accepted methods are also used to detect Expressed Sequence Tags (ESTs) and SSR markers derived from EST sequences, as well as Randomly Amplified Polymorphic DNA (RAPD).
"germplasm" refers to genetic material that belongs to or is derived from an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety, or family), or from a clone of a line, variety, species, or culture, or, more generally, all individuals of a species or species (e.g., maize germplasm collection (maize germplasm collection) or an Andean germplasm collection (Andean germplasm collection)). The germplasm may be part of an organism or cell, or may be isolated from the organism or cell. In general, germplasm provides genetic material with a specific molecular constitution that provides a physical basis for some or all of the genetic qualities of an organism or cell culture. As used herein, germplasm includes cells, seeds, or tissues from which new plants may be grown, or plant parts, such as leaves, stems, pollen, or cells, that may be cultured into whole plants.
A "haplotype" is the genotype, i.e., a combination of alleles, of an individual at multiple genetic loci. Typically, the genetic loci described by the haplotypes are physically and genetically linked, i.e., on the same chromosomal segment.
The term "heterogeneity" is used to indicate that individuals within the cohort differ in genotype at one or more specific loci.
The heterotic response or "heterosis" of a material can be defined by the performance of the average over the parent (or high parent) when crossed with other non-similar or unrelated groups.
The "heterotic group" includes a group of genotypes which perform well when crossed with genotypes from different heterotic groups (Hallauer et al, (1998) Corn Breeding [ maize breeding ], pp. 463-564, in G.F.Sprague and J.W.Dudley editions, Corn and Corn improvement [ maize and maize improvement ]). Inbred lines are divided into a dominant group of hybrids and further subdivided into families in the dominant group of hybrids based on several criteria such as pedigree, association based on molecular markers and performance in hybrid combinations (Smith et al, (1990) the or. appl. Gen. [ theories and applied genetics ] 80: 833-. In the united states, the two most widely used heterotic groups are known as the "Iowa stilf stage Synthetic" (also referred to herein as "rigid stalks") and the "Lancaster" (Lancaster) or "Lancaster Sure Crop" (sometimes referred to as NSS or non-rigid stalks).
Some heterosis groups possess the traits required to become the female parent, and others possess the traits required to become the male parent. For example, in maize, the yield results from the release of public inbred lines from a population called BSSS (iowa rigid stalk synthetic population) have led these inbred lines and their derivatives to be a female pool in the middle maize band. The BSSS inbred line has been crossed with other inbred lines (e.g., SD 105 and maize amanba (maize Amargo)), and a general group of this material has been known as rigid Stalk synthesis (SSS), even though not all inbreds are derived from the original BSSS population (Mikel and Dudley, (2006) Crop science: 46: 1193-. By default, all other inbreds that bind well to the SSS inbred are assigned to the male pool, named NSS, i.e. non-rigid stalk, due to lack of better name. This group includes several major heterotic groups, such as the lanchester harvest (Lancaster surerop), indion (Iodent) and rimming maize (Leaming Corn).
The term "homogeneity" means that the members of a group have the same genotype at one or more specific loci.
The term "hybrid" refers to the progeny obtained between crosses of at least two genetically distinct parents.
The term "inbred line" refers to a line that has been bred to obtain genetic homogeneity.
The term "indel" refers to an insertion or deletion, wherein one line may be referred to as a nucleotide or DNA fragment having an insertion relative to a second line, or the second line may be referred to as a nucleotide or DNA fragment having a deletion relative to the first line.
The term "introgression" refers to the phenomenon of the transmission of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired allele at a given locus can be transmitted to at least one progeny via sexual crossing between two parents of the same species, wherein at least one of the parents has the desired allele within its genome. Alternatively, for example, the transmission of the allele can occur by recombination between two donor genomes, for example in fusion protoplasts, wherein at least one of the donor protoplasts has the desired allele in its genome. The desired allele can be detected at the QTL, transgene, etc., for example, by a marker associated with the phenotype. In any case, progeny comprising the desired allele can be backcrossed repeatedly with lines having the desired genetic background and selected for the desired allele to produce an allele that is fixed in the selected genetic background.
When "introgression" is repeated two or more times, the process is often referred to as "backcrossing".
A "line" or "breed" is a group of individuals with the same parents, which are usually inbred to some extent and are usually homozygous and homogeneous (isogenic or nearly isogenic) at most loci. "sublines" refer to a subpopulation of inbreds that is genetically distinct from other similar subpopulations of inbreds originating from the same ancestor.
As used herein, the term "linkage" is used to describe the degree to which one marker locus is associated with another marker locus or some other locus. Linkage relationships between molecular markers and loci that affect a phenotype are expressed in terms of "probability" or "probability of modulation". Linkage may be expressed as a desired limit or range. For example, in some embodiments, when any marker is in a single meiosis map (based on a population that has undergone a round of meiosis (e.g., F)2) A genetic map of (a); IBM2 map consisting of multiple meioses) are linked (genetically or physically) when less than 50, 40, 30, 25, 20, or 15 map-distance units (or cM) are separated. In some aspects, it is advantageous to define a bracketed linkage range, for example, between 10cM and 20cM, between 10cM and 30cM, or between 10cM and 40 cM. The more tightly the marker is linked to the second locus, the better the marker will indicate the second locus. Thus, a "closely linked locus", e.g., a marker locus and a second locus, exhibits 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, yet more preferably about 4% or less, yet more preferably about 3% or less, toAnd still more preferably an interlocus recombination frequency of about 2% or less. In highly preferred embodiments, the relevant loci exhibit a recombination frequency of about 1% or less, for example about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or less. Two loci that are located on the same chromosome and have a distance such that recombination between the two loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25% or less) are also considered to be "adjacent" to each other. Since one cM is the distance between two markers showing a recombination frequency of 1%, any marker is tightly linked (both genetically and physically) to any other marker in close proximity (e.g., at a distance equal to or less than 10 cM). Two closely linked markers on the same chromosome may be located 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5 or 0.25cM or less from each other.
The term "linkage disequilibrium" refers to the non-random segregation of genetic loci or traits (or both). In either case, linkage disequilibrium means that the relevant loci are physically close enough along a stretch of chromosome that they separate together with a higher frequency than random (i.e., non-random). Markers that show linkage disequilibrium are considered linked. Linked loci have more than 50% chance (e.g., about 51% to about 100% chance) of co-segregating. In other words, two markers that co-segregate have a recombination frequency of less than 50% (and by definition, less than 50cM apart on the same linkage group). As used herein, linkage may exist between two markers, or alternatively, between a marker and a locus that affects a phenotype. A marker locus may be "associated with" (linked to) a trait. The degree of linkage of a marker locus to a locus affecting a phenotypic trait is measured, for example, by the statistical probability (e.g., F statistics or LOD score) that the molecular marker cosegregates with the phenotype.
Linkage disequilibrium is most commonly measured by the metric r2Evaluation, said measure r2Calculated using the formula in the following literature: hill, w.g. and Robertson, a, the or]38: 226-231(1968). When r is2The presence of a complete LD between two marker loci when 1 means that the markers have not been recombinantly isolated and have the same allele frequency. Said r2The values depend on the population used. r is2A value greater than 1/3 shows a strong enough LD for localization (Ardlie et al, Nature Reviews Genetics [ Nature Reviews by Nature Reviews)]3: 299-309(2002)). Thus, r between marker loci as a pair2An allele is in linkage disequilibrium at a value greater than or equal to 0.33, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.
As used herein, "linkage equilibrium" describes a situation in which two markers segregate independently, i.e., are randomly assigned among offspring. Markers that show linkage equilibrium are considered unlinked (whether they are located on the same chromosome or not).
A "locus" is a location on a chromosome, e.g., where a nucleotide, gene, sequence, or marker is located.
"LOD value" or "LOD score" (Risch, Science 255: 803-. An LOD score between two markers of three indicates a probability of linkage 1000 times higher than the probability of no linkage, and an LOD score of two indicates a probability of linkage 100 times higher than the probability of no linkage. A LOD score greater than or equal to two may be used to detect linkage. The LOD score can also be used to show the strength of association between a marker locus and a quantitative trait in a "quantitative trait locus" mapping. In this case, the magnitude of the LOD score depends on the closeness between the marker locus and the locus affecting the quantitative trait, as well as the magnitude of the quantitative trait effect.
The term "plant" includes whole plants, plant cells, plant protoplasts, plant cell or tissue cultures from which plants can be regenerated, plant calli, plant clumps and whole plant cells or plant parts in plants, such as seeds, flowers, cotyledons, leaves, stems, shoots, roots, root tips and the like. As used herein, "modified plant" means any plant that has a genetic change due to human intervention. The modified plant may have genetic changes introduced by plant transformation, genome editing, or conventional plant breeding.
A "marker" is a means of finding a position on a genetic or physical map or of finding a linkage between a marker and a trait locus (a locus that affects a trait). The location at which the marker is detected can be known by detecting the polymorphic allele and its genetic location, or by hybridizing, sequence matching or amplifying sequences that have been physically mapped. The marker may be a DNA marker (detecting DNA polymorphisms), a protein (detecting variations in the encoded polypeptide), or a simple inherited phenotype (such as a "wax" phenotype). DNA tags can be developed from genomic nucleotide sequences or from expressed nucleotide sequences (e.g., from spliced RNA or cDNA). According to the DNA marker technique, the marker consists of complementary primers flanking the locus and/or complementary probes hybridizing to polymorphic alleles at the locus. A DNA marker or genetic marker may also be used to describe a gene, DNA sequence or nucleotide on the chromosome itself (rather than to detect components of the gene or DNA sequence), and is typically used when the DNA marker is associated with a particular trait in human genetics (e.g., a breast cancer marker). The term marker locus is the locus (gene, sequence or nucleotide) at which the marker is detected.
Markers for detecting genetic polymorphisms between members of a population are well known in the art. The marker may be defined by the type of polymorphism it detects and the labeling technique used to detect the polymorphism. Types of markers include, but are not limited to: restriction fragment length polymorphism detection (RFLP), isozyme marker detection, randomly amplified polymorphic dna (rapd), amplified fragment length polymorphism detection (AFLP), simple repeat sequence detection (SSR), amplified variable sequence detection of plant genomes, autonomous sequence replication detection, or single nucleotide polymorphism detection (SNP). SNPs can be detected, for example, by DNA sequencing, PCR-based sequence-specific amplification methods, polynucleotide polymorphism detection by allele-specific hybridization (ASH), dynamic allele-specific hybridization (DASH), molecular beacons, microarray hybridization, oligonucleotide ligase analysis, Flap endonuclease, 5' endonuclease, primer extension, single-strand conformation polymorphism (SSCP), or Temperature Gradient Gel Electrophoresis (TGGE). DNA sequencing, such as pyrosequencing techniques, has the advantage of being able to detect a series of linked SNP alleles that make up a haplotype. Haplotypes tend to be more informative (detect higher levels of polymorphism) than SNPs.
A "marker allele," alternatively an "allele of a marker locus," can refer to one of a plurality of polymorphic nucleotide sequences found at a marker locus in a population.
"marker assisted selection" (MAS) is a method for selecting individual plants based on marker genotype.
"marker assisted counter-selection" is a method whereby a marker genotype is used to identify plants that will not be selected, such that the plants are removed from breeding programs or planting.
"marker haplotype" refers to a combination of alleles at a marker locus.
A "marker locus" is a specific chromosomal location in the genome of a species at which a specific marker can be found. The marker locus can be used to track the presence of a second linked locus (e.g., a linked locus that affects the expression of a phenotypic trait). For example, marker loci can be used to monitor segregation of alleles at genetically or physically linked loci.
As noted above, the term "molecular marker" may be used to refer to a genetic marker, or an encoded product (e.g., a protein) that is used as a point of reference when identifying linked loci. The tag can be derived from a genomic nucleotide sequence or from an expressed nucleotide sequence (e.g., from spliced RNA, cDNA, etc.), or from an encoded polypeptide. The term also refers to nucleic acid sequences complementary to or flanking the marker sequence, 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 or absence of a marker locus, e.g., a nucleic acid probe that is complementary to a marker locus sequence. Alternatively, in certain aspects, a marker probe refers to any type of probe (i.e., genotype) that is capable of distinguishing between particular alleles present at a marker locus. Nucleic acids are "complementary" when they specifically hybridize in solution. When located in an indel region, such as the non-collinear regions described herein, some of the markers described herein are also referred to as hybridization markers. This is because, by definition, the insertion region is a polymorphism with respect to a plant that does not have the insertion. Thus, the marker need only indicate whether the indel region is present. Any suitable marker detection technique may be used to identify such hybridization markers, for example using SNP techniques in the examples provided herein.
An allele is "negatively" associated with a trait when the allele is linked to the trait, and when the presence of the allele is an indication that the desired trait or trait form will not be present in a plant comprising the allele.
The terms "phenotype", "phenotypic trait" or "trait" may refer to observable expression of a gene or series of genes. The phenotype may be observable to the naked eye, or by any other means of assessment known in the art (e.g., weighing, counting, measuring (length, width, angle, etc.), microscopy, biochemical analysis, or electromechanical assay). In some cases, the phenotype is directly controlled by a single gene or genetic locus, i.e., a "monogenic trait" or a "simple genetic trait". In the absence of large levels of environmental changes, monogenic traits may segregate in a population to give a "mass" or "discrete" distribution, i.e., the phenotype falls into a discrete category. In other cases, a phenotype is the result of multiple genes and can be considered a "polygenic trait" or a "complex trait. The polygenic traits segregate in a population to give a "quantitative" or "continuous" distribution, i.e., the phenotypes cannot segregate into discrete classes. Both monogenic and polygenic traits may be affected by the environment in which they are expressed, but polygenic traits tend to have a greater environmental component.
A "physical map" of a genome is a map that shows the linear order of identifiable markers (including genes, markers, etc.) on chromosomal DNA. However, in contrast to genetic maps, the distance between markers is absolute (e.g., measured in base pairs or separate and overlapping contiguous gene segments) and is not based on genetic recombination (which may vary in different populations).
A "polymorphism" is a variation in DNA between 2 or more individuals within a population. The polymorphism preferably has a frequency of at least 1% in the population. Useful polymorphisms may include Single Nucleotide Polymorphisms (SNPs), simple repeat sequences (SSRs), or insertion/deletion polymorphisms (also referred to herein as "indels").
"production marker" or "production SNP marker" is a marker that has been developed for high throughput purposes. Production of SNP markers was developed for the detection of specific polymorphisms and designed for use with a variety of chemical reactions and platforms.
The term "quantitative trait locus" or "QTL" refers to a region of DNA associated with differential expression of a quantitative phenotypic trait in at least one genetic background (e.g., in at least one breeding population). A region of a QTL encompasses or is closely linked to one or more genes affecting the trait in question.
A "reference sequence" or "consensus sequence" is a defined sequence that is used as a basis for sequence alignment. The labeled reference sequence is obtained by sequencing multiple lines at that locus, aligning the nucleotide sequences in a sequence alignment program (e.g., Sequencher) and then obtaining the most common nucleotide sequence for the alignment. Polymorphisms found in these individual sequences are annotated in the consensus sequence. The reference sequence is typically not an exact copy of any individual DNA sequence, but rather represents a mixture of available sequences and is used to design primers and probes for polymorphisms within that sequence.
An "unfavorable allele" of a marker is one that segregates with the unfavorable plant phenotype, thus providing the benefit of identifying plants that can be removed from breeding programs or planting.
The term "yield" refers to the productivity per unit area of a particular plant product of commercial value. Yield is affected by both genetic and environmental factors. "agronomic", "agronomic trait", and "agronomic trait performance" refer to a trait (and potentially genetic elements) of a given plant variety that contributes to yield during the growing period. Individual agronomic traits include emergence vigour, stress tolerance, disease resistance or tolerance, herbicide resistance, branching, flowering, seed formation, seed size, seed density, lodging resistance, threshing performance and the like. Yield is thus the final vertex of all agronomic traits.
Provided herein are marker loci that exhibit statistically significant co-segregation with disease resistance traits that confer broad resistance to one or more specific diseases. Detection of these loci or additional linked loci, as well as resistance genes, can be used in marker assisted selection as part of breeding programs to produce plants that are resistant to one or more diseases.
It has been recognized that in quite some cases, specific genetic loci associated with a specific phenotype (e.g., disease resistance) can be located in the genome of an organism. Plant breeders can advantageously use molecular markers to identify desired individuals by detecting marker alleles that exhibit statistically significant probabilities of co-segregating with a desired phenotype, as evidenced by linkage disequilibrium. By identifying molecular markers or clusters of molecular markers that co-segregate with traits of interest, the plant breeder is able to rapidly select for a desired phenotype by selecting the appropriate molecular marker alleles (a process known as marker assisted selection or MAS).
Various methods known in the art can be used to detect molecular markers or clusters of molecular markers that co-segregate with a trait of interest (e.g., a disease resistance trait). The basic idea behind these methods is to detect markers of alternative genotypes (or alleles) with significantly different average phenotypes. Thus, the magnitude of the difference, or the level of significance of the difference, between alternative genotypes (or alleles) between marker loci is compared. Inferring that the trait gene is located closest to the one or more markers having the greatest relatedness for the genotype difference. Two such methods for detecting a trait locus of interest are: 1) population-based association analysis (i.e., association mapping) and 2) traditional linkage analysis.
Associative positioning
Understanding the degree and pattern of Linkage Disequilibrium (LD) in the genome is a prerequisite for the development of efficient, associative methods to identify and map Quantitative Trait Loci (QTLs). Linkage Disequilibrium (LD) refers to the non-random association of alleles in a collection of individuals. When LD is observed in alleles at linked loci, LD is measured as the attenuation of LD across a specific region of the chromosome. The range of LD reflects the recombination history of this region. The average rate of LD decay in the genome can help predict the number and density of markers needed to perform genome-wide association studies and provide an estimate of the resolution that can be expected.
The association or LD localization is aimed at identifying significant genotype-phenotype associations. It has been developed and utilized as a powerful tool for fine positioning in the cross-breeding of: human (Corder et al, (1994) "Protective effect of protein-E type-2 alloy for late-on Alzheimer's disease" protection of apolipoprotein E2 type allele against late-type Alzheimer's disease, Nat Genet [ Nature genetics ] 7: 180-184; Hastbackka et al, (1992) "Linkage disequilibrium mapping expressed in genes: Diastric dyssplasia in Finland ] Nat Genet [ natural genetics ] 2: 204-211; Kerem et al, (1989)" Linkage disequilibrium of genes: genetic analysis [ Nature genetics ] 2: 204-211; Kerem et al, (1989) "scientific" Linkage disequilibrium of genes: genetic analysis [ Nature genetics ] 1080 ] and maize 3: 1073-linked genes of maize [ maize ] 2: 1073, and Zeolite, proc Natl Acad Sci USA [ Proc Natl Acad Sci USA ] 98: 11479-11484; thornsberry et al, (2001) "Dwarf 8 polymorphisms associated with variation in flowering time [ Dwarf8 polymorphism ] Nat Genet [ Nat genetics ] 28: 286-; reviewed by Flint-Garcia et al, (2003) "Structure of linkage disequilibrium in plants", Annu Rev Plant Biol. [ Plant biological evaluation ] 54: 357, 374) where recombination between heterozygotes is frequent and results in rapid decay of LD. In inbred species, recombination between homozygous genotypes is not genetically detectable, the degree of LD is greater (i.e., larger linked marker blocks are inherited together) and this greatly improves the detectability of linkage localization (Wall and Pritcard, (2003) "Haplotpype blocks and linkage disequilibrium in the human genome [ Haplotype blocks and linkage disequilibrium in the human genome ]", Nat Rev Genet [ Nature genetics review ] 4: 587-.
The recombination and mutation history of the population is a function of mating habits and the effective size and age of the population. Larger population sizes provide enhanced possibilities for detecting recombination, while older populations are often associated with higher levels of polymorphism, both of which contribute to the observed significant increase in LD decay rate. On the other hand, smaller effective population sizes, such as those that have experienced recent genetic bottlenecks, tend to exhibit slower rates of LD decay, leading to broader haplotype conservation (Flint-Garcia et al, (2003) "Structure of linkage disequilibrium in plants", Annu Rev Plant Biol. [ Plant biology review ] 54: 357-.
Good breeding lines provide a valuable starting point for association analysis. Correlation analysis quantitative phenotype scores (e.g., disease tolerance grades from one to nine for each line) were used in the analysis (rather than considering only the tolerance and resistance allele frequency distributions in the analysis of the inter-group allele distribution types). The availability of detailed phenotypic performance data collected through breeding programs and the environment of a large number of elite lines over the years provides a valuable data set for genetic marker association mapping analysis. This paves the way for seamless integration between research and applications, and takes advantage of historically accumulated data sets. However, understanding the relationship between polymorphisms and recombination is useful for developing appropriate strategies for efficiently extracting the maximum information from these resources.
This type of correlation analysis neither produces nor requires any map data, but is independent of map location. This analysis compares the phenotypic score of the plant with the genotype at different loci. Subsequently, using previously determined map locations for these markers, any suitable map (e.g., a composite map) can optionally be used to aid in the observation of the distribution of the identified QTL markers and/or QTL marker clusters.
Classical linkage analysis
Traditional linkage analysis is based on the same principle; however, LDs are generated by creating populations from a small number of founders. The creator is selected to maximize the level of polymorphism within the structured population and to assess the level of co-segregation of the polymorphic site with a given phenotype. A number of statistical methods have been used to identify significant marker-trait associations. One such method is the interval localization method (Lander and Botstein, Genetics [ Genetics ] 121: 185-199(1989) in which each of a number of positions along a genetic map (say an interval of 1 cM) is tested for the probability that the gene controlling the trait of interest is located at that position.
Provided herein are marker loci that exhibit statistically significant co-segregation with disease resistance traits as determined by traditional linkage analysis and genome-wide association analysis. Detection of these loci or additional linked loci can be used in marker assisted breeding programs to produce plants with disease resistance.
Activities in marker assisted breeding programs may include, but are not limited to: selecting among new breeding populations based on historical genotype and agronomic trait associations to identify which population has the highest frequency of favorable nucleic acid sequences, selecting among progeny in breeding populations for favorable nucleic acid sequences, selecting among parental lines based on prediction of progeny performance, and advancing lines in germplasm improvement activities based on the presence of favorable nucleic acid sequences.
Chromosomal intervals associated with disease resistance traits are provided. A variety of methods well known in the art can be used to identify chromosomal intervals. The boundaries of such chromosomal intervals are extended to encompass markers that will be linked to one or more genes that control the trait of interest. In other words, the chromosomal interval is extended such that any marker located within the interval (including the end markers that define the boundaries of the interval) can be used as a marker for the disease resistance trait.
Conversely, if, for example, two markers in close proximity show co-segregation with the desired phenotypic trait, it is sometimes unclear whether each of those markers identifies the same gene or two different genes or multiple genes. In any event, knowledge of how many genes are within a particular physical/genomic interval is not necessary to formulate or practice which is presented in this disclosure.
Chromosome 7 interval can comprise any marker identified herein as being associated with a disease resistance trait comprising a sequence that is identical to SEQ ID NO: 4-10 or 13-16, having at least 95% identity. Any marker located within these intervals can be used as a marker for disease resistance and can be used in the context of the methods presented herein to identify and/or select plants that have disease resistance, whether or not the resistance is newly conferred or enhanced compared to control plants. In certain embodiments, markers located upstream and downstream of the ZmMM1 gene location are genetically and physically very closely linked and thus can be used to select the ZmMM1 gene for trait introgression and product development.
The chromosomal interval may also be defined by a marker linked to a disease resistance gene (which exhibits linkage disequilibrium therewith), and r2Is a common measure of Linkage Disequilibrium (LD) in the context of relevance studies. If r of LD between chromosome 7 marker locus and another immediately adjacent chromosome 7 marker locus in the interval of interest2A value of greater than 1/3(Ardlie et al, Nature Reviews Genetics [ Nature Reviews by Nature]3: 299-309(2002)), the two loci are in linkage disequilibrium with each other.
A common measure of linkage is the frequency of co-segregation of traits. This can be expressed as a percentage of co-segregation (recombination frequency), or expressed in centimorgans (cM). cM is a measure of the frequency of genetic recombination. One cM equals 1% chance that a trait at one genetic locus will segregate from a trait at another locus due to hybridization in a single generation (meaning that these traits all share 99% of the chance to segregate). Since chromosomal distance is roughly proportional to the frequency of hybridization events between traits, there is an approximate physical distance associated with recombination frequency.
The marker locus itself is a trait and can be assessed during segregation by following the marker locus according to standard linkage analysis. Thus, one cM equals 1% chance that one marker locus will segregate from another due to hybridization in a single generation.
The closer a marker is to a gene controlling a trait of interest, the more efficient and advantageous the marker is as an indication of the desired trait. Closely linked loci exhibit an interlocus hybridization frequency of about 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, still more preferably about 4% or less, yet more preferably about 3% or less, and still more preferably about 2% or less. In highly preferred embodiments, the relevant loci (e.g., marker locus and target locus) exhibit a recombination frequency of about 1% or less, such as about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or less. Thus, the loci are separated by a distance of about 10cM, 9cM, 8cM, 7cM, 6cM, 5cM, 4cM, 3cM, 2cM, 1cM, 0.75cM, 0.5cM, or 0.25cM or less. In other words, two loci that are located on the same chromosome and have a distance such that recombination between the two loci occurs with a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25% or less) are considered "adjacent" to each other.
Although a particular marker allele may co-segregate with a disease resistance trait, it is important to note that the marker locus does not necessarily result in expression of the disease resistance phenotype. For example, it is not a requirement that the marker polynucleotide sequence be part of a gene that produces a disease resistance phenotype (e.g., be part of the open reading frame of a gene). The association between a specific marker allele and a disease resistance trait is due to an initial "coupling" linkage between the marker allele and the allele in the ancestral line from which the allele originates. Finally, by repeated recombination, hybridization events between the marker and the genetic locus can alter this orientation. For this reason, the favorable marker allele can be altered according to the linkage phase present in the disease-resistant parent used to create the segregating population. This does not alter the fact that the markers can be used to monitor phenotypic segregation. It merely changes which marker allele is considered advantageous in a given segregating population.
The methods presented herein comprise detecting the presence of one or more marker alleles associated with disease resistance in a plant, and then identifying and/or selecting plants that have favorable alleles at those marker loci. Markers have been identified herein as being associated with disease resistance traits and can therefore be used to predict disease resistance in plants. Any marker within 50cM, 40cM, 30cM, 20cM, 15cM, 10cM, 9cM, 8cM, 7cM, 6cM, 5cM, 4cM, 3cM, 2cM, 1cM, 0.75cM, 0.5cM or 0.25cM (based on the genetic map of a single meiosis) may also be used to predict disease resistance in plants.
The Molecular markers can be used in a variety of Plant breeding applications (see, e.g., Staub et al, (1996) Hortsccience [ horticulture ] 31: 729-. One of the main areas of interest is the use of Marker Assisted Selection (MAS) to increase the efficiency of backcrossing and introgression. Molecular markers that exhibit linkage to loci that affect a desired phenotypic trait provide a useful tool for selecting traits in a plant population. This is particularly true where the phenotype is difficult to determine. Since DNA marker assays are more labor-efficient and occupy less physical space than field phenotypic analysis, larger populations can be assayed, increasing the probability of finding recombinants with target segments that move from donor lines to recipient lines. The closer the linkage, the more useful the marker is because recombination is less likely to occur between the marker and the gene causing the trait, which could lead to false positives. Flanking markers reduces the probability of false positive selection occurring due to the need for double recombination events. It is desirable that the gene itself has a marker so that recombination between the marker and the gene cannot occur. In some embodiments, the methods disclosed herein generate markers in disease resistance genes, wherein the genes are identified by inferring genomic location from clustering or cluster analysis of conserved domains.
When the gene is introgressed by MAS, not only the gene but also the flanking regions are introduced (Gepts. (2002). Crop Sci [ Crop science ]; 42: 1780-. This is called "linkage drag". In the case where the donor plant is not very related to the recipient plant, these flanking regions carry additional genes which may encode agronomically undesirable traits. This "linkage drag" can result in yield loss or other negative agronomic characteristics even after backcrossing with elite lines for many cycles. This is sometimes referred to as "yield drag". The size of the flanking regions can be reduced by additional backcrossing, although this is not always successful because the breeder cannot control the size of the region or recombination breakpoint (Young et al, (1998) Genetics 120: 579-. In classical breeding, recombination which contributes to the reduction of the size of the donor segment is usually chosen only by chance (Tanksley et al, (1989). Biotechnology [ Biotechnology ] 7: 257-. Even after 20 backcrosses of this type, it is expected that a considerable fragment of the donor chromosome still linked to the gene will be found to be selected. However, if a marker is used, it is possible to select rare individuals that have undergone recombination in the vicinity of the gene of interest. Of the 150 backcross plants, there is a 95% chance that at least one plant will undergo a cross within 1cM (based on single meiosis map distance) of the gene. The markers enable unambiguous identification of these individuals. With one additional backcross of 300 plants, there was a 95% probability of crossing within 1cM single meiosis pattern distance on the other side of the gene, resulting in a segment near the target gene of less than 2cM based on single meiosis pattern distance. This can be achieved in two generations with labeling, whereas an average of 100 generations would be required without labeling (see Tanksley et al, supra). When the exact location of a gene is known, flanking markers surrounding the gene can be used to select for recombination in different population sizes. For example, in a smaller population, it is expected that recombination may be further away from the gene, thus requiring more distal flanking markers to detect the recombination.
The main components for implementing MAS are: (i) defining a population in which marker-trait associations are to be determined, which may be a separate population, or a random or structured population; (ii) monitoring the segregation or association of the polymorphic markers relative to the trait and determining linkage or association using statistical methods; (iii) (iii) defining a set of desired markers based on the results of the statistical analysis, and (iv) using and/or extrapolating this information into the current breeding germplasm set to enable marker-based selection decisions to be made. The markers described in this disclosure, as well as other marker types, such as SSR and FLP, can be used in marker-assisted selection schemes.
SSRs can be defined as relatively short runs of tandem repeat DNA of 6bp or less in length (Tautz (1989) Nucleic Acid Research [ Nucleic Acid Research ] 17: 6463-6471; Wang et al (1994) the national and Applied Genetics [ theory and Applied Genetics ], 88: 1-6). Polymorphisms arise due to changes in the number of repeat units, which may be caused by slippage during DNA replication (Levinson and Gutman (1987) Mol Biol Evol [ molecular biology and evolution ] 4: 203-. Changes in repeat length can be detected by designing PCR primers to conserved non-repetitive flanking regions (Weber and May (1989) Am J Hum Genet. [ human genetics in USA ] 44: 388-. Since SSRs are multiallelic, co-dominant, reproducible and suitable for high-throughput automation, they are very suitable for mapping and MAS (Rafalski et al, (1996) Generation and use of DNA markers in plants [ DNA markers are generated and used in plants ] in Non-mammalian genomic analysis: a practical guide [ Non-mammalian genomic analysis: practical guide ]. Academic Press, pp.75-135).
Various types of SSR markers can be produced, and SSR spectra can be obtained by gel electrophoresis of the amplified products. The score for the marker genotype is based on the size of the amplified fragment.
Various types of FLP markers may also be generated. Most commonly, amplification primers are used to generate fragment length polymorphisms. Such FLP markers are similar in many respects to SSR markers, except that the region amplified by the primers is generally not a highly repetitive region. The amplified region or amplicon is still sufficiently variable between germplasms, usually due to insertions or deletions, to enable fragments generated by the amplification primers to be distinguished in polymorphic individuals, and it is known that such insertions often occur in maize (Bhattramakki et al, (2002).
SNP markers detect single base pair nucleotide substitutions. Among all Molecular marker types, SNPs are the most abundant and therefore have the potential to provide the highest genetic map resolution (Bhattramakki et al, 2002Plant Molecular Biology]48: 539-547). Since SNPs do not require large amounts of DNA and automation of the assay can be straightforward, SNPs can be assayed in a so-called "ultra-high-throughput" manner, at even higher throughput levels than SSRs. SNPs are also likely to be relatively low cost systems. These three factors together make the use of SNPs in MAS highly attractive. Several methods are available for SNP genotyping, including but not limited to: hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, micro-sequencing and coded spheres (coded spheres). These methods have been reviewed in the following documents: gut (2001) Hum Mutat [ human Gene mutation]17, pages 475-492; shi (2001) Clin Chem [ clinical chemistry ]]47, page 164-172; kwok (2000) Pharmacogenomics 1[ Pharmacogenomics 1 ]]Pages 95-100; and Bhattramakki and Rafalski (2001), Discovery and application of single nucleotide polymorphism markers in plants]At the following stage: henry, edited, Plant Genotyping: the DNA converting of Plants, CABI Publishing, Wallingford [ plant genotyping: DNA fingerprinting of plants, CABI Press, Walinford]In (1). These and other methods are utilized by a wide range of commercially available techniques for detecting SNPs, including: masscode. tm. (Qiagen corporation),
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A number of SNPs within or across a linkage sequence can be used to describe haplotypes for any particular genotype (Ching et al, (2002), BMC Genet, [ BMC genetics ] 3: 19, Gupta et al, 2001, Rafalski (2002b), Plant Science [ Plant Science ] 162: 329-. Haplotypes can be more informative than a single SNP, and any particular genotype can be described in more detail. For example, a single SNP may be an allele "T" of a particular line or variety with disease resistance, but the allele "T" may also occur in a breeding population used to recurrent parents. In this case, the haplotype (e.g., the combination of alleles at linked SNP markers) may be more informative. Once a unique haplotype is assigned to a donor chromosomal region, that haplotype can be used in that population, or any subset thereof, to determine whether an individual has a particular gene. See, for example, WO 2003054229. The use of automated high-throughput label detection platforms known to those of ordinary skill in the art makes this method efficient and effective.
Many of the markers set forth herein can be readily used as Single Nucleotide Polymorphism (SNP) markers to select the ZmMM1 gene. Primers are used to amplify DNA segments of individuals (preferably inbred lines) representing the diversity of the population of interest using PCR. The PCR products were sequenced directly in one or both directions. The resulting sequences are aligned and polymorphisms identified. The polymorphisms are not limited to Single Nucleotide Polymorphisms (SNPs), but include indels, CAPS, SSRs, and VNTRs (variable number of tandem repeats). In particular, one can readily use the information provided herein to obtain additional polymorphic SNPs (and other markers) within the region amplified by the primers disclosed herein for the fine map information described herein. Markers within the described map regions can be hybridized to BACs or other genomic libraries, or electronically aligned with genomic sequences to find new sequences in the same approximate location as the marker.
In addition to the SSR, FLP, and SNP described above, other types of molecular markers are also widely used, including but not limited to: expressed Sequence Tags (ESTs), SSR markers derived from EST sequences, Randomly Amplified Polymorphic DNA (RAPD), and other nucleic acid-based markers.
The isozyme spectrum and the linked morphological characteristics may also be used indirectly as markers in some cases. Although they do not detect DNA differences directly, they are often affected by specific genetic differences. However, the markers for detecting DNA variation are much more numerous and polymorphic than isozymes or morphological markers (Tanksley (1983) Plant Molecular Biology Reporter: [ Plant Molecular Biology guide ] 1: 3-8).
Sequence alignments or contigs can also be used to find sequences upstream or downstream of the specific markers listed herein. These new sequences, close to the markers described herein, are then used to find and develop functionally equivalent markers. For example, different physical and/or genetic maps are aligned to locate equivalent markers not described in this disclosure but located within similar regions. These maps may be within a species, or even across other species that are genetically or physically aligned.
Generally, MAS uses polymorphic markers that have been identified as having a significant likelihood of co-segregating with traits such as the SCR disease resistance trait. Such markers are presumed to be located on the map in the vicinity of one or more genes conferring a disease resistance phenotype on a plant and are considered indicative of a desired trait or marker. Plants are tested for the presence of a desired allele in the marker, and plants containing the desired genotype at one or more loci are expected to transfer the desired genotype, along with the desired phenotype, to their progeny. Thus, plants that are resistant to SCR disease can be selected by detecting one or more marker alleles, and in addition, progeny plants from these plants can be selected. Thus, a plant containing the desired genotype (i.e., the genotype associated with disease resistance) in a given chromosomal region is obtained and then crossed with another plant. Progeny of such crosses are then genotypically evaluated using one or more markers, and progeny plants having the same genotype in a given chromosomal region are then selected for disease resistance.
SNPs (i.e., SNP haplotypes) can be used alone or in combination to select favorable resistance gene alleles associated with disease resistance. For example, the SNP haplotype on chromosome 7 QTL comprises a sequence identical to SEQ ID NO: 4-10 or 13-16, any SNPs or indels are as shown in figure 1, or combinations thereof.
One skilled in the art would expect that additional polymorphic sites may be present at marker loci in and near the chromosomal markers identified by the methods disclosed herein, where one or more of the polymorphic sites are in Linkage Disequilibrium (LD) with an allele at one or more of the polymorphic sites in the haplotype, and thus may be used in marker assisted selection procedures to introgress the allele of interest or genomic fragment of interest. Two particular alleles at different polymorphic sites are considered to be in LD if the presence of an allele at one of these sites tends to predict the presence of alleles at other sites on the same chromosome (Stevens, mol. Diag. [ molecular diagnostics ] 4: 309-17 (1999)). The marker locus may be located within 5cM, 2cM, or 1cM of the disease resistance trait QTL (on a single meiosis based genetic map).
The skilled person will appreciate that allele frequencies (and thus haplotype frequencies) may differ from germplasm pool to germplasm pool. Germplasm inventory varies due to differences in maturity, heterosis grouping, geographic distribution, and the like. Thus, SNPs and other polymorphisms in certain germplasm pools may not be informative.
Plants identified, modified and/or selected by any of the above methods are also of interest.
The present disclosure encompasses ZmMM1 polypeptides. As used herein, "ZmMM 1 polypeptide" and "ZmMM 1 protein" are used interchangeably and refer to one or more polypeptides having disease resistance activity and which are complementary to SEQ ID NO: 1-3 is substantially identical to the ZmMM1 polypeptide. A variety of ZmMM1 polypeptides are contemplated.
"substantially identical" as used herein refers to amino acid sequences having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. In some embodiments, the sequence identity is to the full-length sequence of the polypeptide. The term "about" when used herein with respect to percentage of sequence identity means +/-1.0%.
"recombinant protein" as used herein refers to a protein that is no longer in its natural environment (e.g., in vitro or in a recombinant bacterial or plant host cell); a protein expressed from a polynucleotide which has been edited from its native version; or a protein expressed from a polynucleotide at a different genomic position relative to the native sequence.
As used herein, "substantially free of cellular material" refers to polypeptides comprising a protein preparation having less than about 30%, 20%, 10%, or 5% (by dry weight) of non-target proteins (also referred to herein as "contaminating proteins").
"fragments" or "biologically active portions" include polypeptide fragments or polynucleotide fragments that comprise a sequence substantially identical to the ZmMM1 gene polypeptide or polynucleotide, respectively, and that exhibit disease resistance when expressed in a plant.
As used herein, a "variant" refers to a protein or polypeptide having an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a parent amino acid sequence.
In some embodiments, the ZmMM1 polypeptide comprises a sequence identical to SEQ ID NO: 1-3, or a fragment thereof, having at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, wherein the zmm 1 polypeptide has disease resistance when expressed in a plant.
Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the ZmMM1 polypeptide may be prepared by mutations in the DNA. This can also be accomplished by one of several mutagenic forms, like for example site-specific double-strand-break techniques, and/or directed evolution. In some aspects, the encoded change in the amino acid sequence will not substantially affect the function of the protein. Such variants will have the desired activity. However, it is understood that the ability of the ZmMM1 polypeptide to confer disease resistance can be improved by using these techniques on the compositions of the present disclosure.
Isolated or recombinant nucleic acid molecules comprising a nucleic acid sequence encoding a ZmMM1 polypeptide, or a biologically active portion thereof, are provided, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding proteins having regions of sequence homology. As used herein, the term "nucleic acid molecule" refers to DNA molecules (e.g., recombinant DNA, cDNA, genomic DNA, plasmid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.
An "isolated" nucleic acid molecule (or DNA), as used herein, refers to a nucleic acid sequence (or DNA) that is no longer in its natural environment (e.g., in vitro). As used herein, a "recombinant" nucleic acid molecule (or DNA) refers to a nucleic acid sequence (or DNA) in a recombinant bacterial or plant host cell; it has been edited from its native sequence; or it may be located at a different position than the native sequence. In some embodiments, an "isolated" or "recombinant" nucleic acid is free of sequences (preferably protein-encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5 'and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For the purposes of this disclosure, "isolated" or "recombinant" when used in reference to a nucleic acid molecule excludes isolated chromosomes. For example, in various embodiments, a recombinant nucleic acid molecule encoding a ZmMM1 polypeptide may comprise less than about 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb, or 0.1kb of nucleic acid sequences that naturally flank the nucleic acid molecule in genomic DNA of a cell derived from the nucleic acid.
In some embodiments, an isolated nucleic acid molecule encoding a ZmMM1 polypeptide has one or more alterations in the nucleic acid sequence compared to a native or genomic nucleic acid sequence. In some embodiments, the alteration of a native or genomic nucleic acid sequence includes, but is not limited to: changes in nucleic acid sequence due to the degeneracy of the genetic code; changes in the nucleic acid sequence due to amino acid substitutions, insertions, deletions and/or additions compared to the native or genomic sequence; removal of one or more introns; a deletion of one or more upstream or downstream regulatory regions; and a deletion of a 5 'and/or 3' untranslated region associated with the genomic nucleic acid sequence. In some embodiments, the nucleic acid molecule encoding the ZmMM1 polypeptide is a non-genomic sequence.
A variety of polynucleotides encoding ZmMM1 polypeptides or related proteins are contemplated. Such polynucleotides, when operably linked to a suitable promoter, transcription termination and/or polyadenylation sequence, can be used to produce a ZmMM1 polypeptide in a host cell. Such polynucleotides may also be used as probes for isolating homologous or substantially homologous polynucleotides encoding a ZmMM1 polypeptide or related protein.
In some embodiments, the nucleic acid molecule encoding the ZmMM1 polypeptide is a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4-10, and variants, fragments, and complements thereof. As used herein, "complementary sequence" refers to a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence such that it can hybridize to the given nucleic acid sequence to form a stable duplex. "variant polynucleotide sequence" as used herein refers to a nucleic acid sequence which encodes the same polypeptide except for the degeneracy of the genetic code.
In some embodiments, the nucleic acid molecule encoding the ZmMM1 polypeptide is a non-genomic nucleic acid sequence. As used herein, a "non-genomic nucleic acid sequence" or "non-genomic nucleic acid molecule" or "non-genomic polynucleotide" refers to a nucleic acid molecule having one or more alterations in the nucleic acid sequence as compared to a native or genomic nucleic acid sequence. In some embodiments, the alteration of a native or genomic nucleic acid molecule includes, but is not limited to: changes in nucleic acid sequence due to the degeneracy of the genetic code; optimization of nucleic acid sequences for expression in plants; a change in the nucleic acid sequence that introduces at least one amino acid substitution, insertion, deletion and/or addition as compared to the native or genomic sequence; removing one or more introns associated with the genomic nucleic acid sequence; inserting one or more heterologous introns; deleting one or more upstream or downstream regulatory regions associated with the genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; deletion of the 5 'and/or 3' untranslated region associated with the genomic nucleic acid sequence; insertion of heterologous 5 'and/or 3' untranslated regions; and modification of polyadenylation sites. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.
In some embodiments, the nucleic acid molecule encoding a ZmMM1 polypeptide disclosed herein is a non-genomic polynucleotide having a nucleotide sequence that is substantially identical to the nucleotide sequence of SEQ ID NO: 4-10 has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, wherein the ZmMM1 polypeptide has disease resistance activity when expressed in a plant.
In some embodiments, the nucleic acid molecule encodes a ZmMM1 polypeptide variant comprising a substitution of the amino acid sequence of SEQ ID NO: 1-3, or a substitution of one or more amino acids of the amino acid sequence of seq id no.
Nucleic acid molecules that are fragments of these nucleic acid sequences encoding the ZmMM1 polypeptide are also encompassed by the embodiments. "fragment" is used herein to refer to a portion of a nucleic acid sequence encoding a ZmMM1 polypeptide. A fragment of the nucleic acid sequence may encode a biologically active portion of the ZmMM1 polypeptide, or it may be a fragment that can be used as a hybridization probe or PCR primer using the methods disclosed below. Nucleic acid molecules that are fragments of a nucleic acid sequence encoding a ZmMM1 polypeptide comprise at least about 150, 180, 210, 240, 270, 300, 330, 360, 400, 450, or 500 consecutive nucleotides or up to the number of nucleotides present in a full-length nucleic acid sequence encoding a ZmMM1 polypeptide identified by the methods disclosed herein, depending on the intended use. As used herein, "contiguous nucleotides" refers to nucleotide residues that are immediately adjacent to each other. Fragments of the nucleic acid sequences of the embodiments will encode protein fragments that retain the biological activity of the ZmMM1 polypeptide and thus retain disease resistance. As used herein, "retain disease resistance" refers to a polypeptide having the sequence of SEQ ID NO: 1-3, at least about 10%, at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95%, or more of the full-length ZmMM1 polypeptide shown therein.
"percent (%) sequence identity" is determined as the percentage of amino acid residues or nucleotides in a candidate sequence (query sequence) that are identical to corresponding amino acid residues or nucleotides in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without regard to any amino acid conservative substitutions as part of the sequence identity, relative to the reference sequence (the subject sequence). Alignments for the purpose of determining percent sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software, such as BLAST, BLAST-2. One skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. The percent identity between two sequences is a function of the number of identical positions common to the sequences (e.g., percent identity for a query sequence-the number of identical positions between the query sequence and the subject sequence/total number of positions for the query sequence x 100).
In some embodiments, the ZmMM1 polynucleotide encodes a polypeptide comprising an amino acid sequence identical to the sequence set forth throughout SEQ ID NO: 1-3, a ZmMM1 polypeptide having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical throughout the length of the amino acid sequence.
The embodiments also encompass nucleic acid molecules encoding variants of the ZmMM1 polypeptide. "variants" of a nucleic acid sequence encoding a ZmMM1 polypeptide include those sequences encoding a ZmMM1 polypeptide identified by the methods disclosed herein, but which differ conservatively due to the degeneracy of the genetic code, as well as those sequences that are substantially identical as described above. Naturally occurring allelic variants can be identified by using well known molecular biology techniques, such as Polymerase Chain Reaction (PCR) and hybridization techniques as outlined below. Variant nucleic acid sequences also include synthetically derived nucleic acid sequences that have been generated, for example, by using site-directed mutagenesis, but still encode the ZmMM1 gene polypeptides disclosed herein.
The skilled artisan will further appreciate that changes may be introduced by mutation of the nucleic acid sequence, resulting in a change in the amino acid sequence of the encoded ZmMM1 polypeptide, without altering the biological activity of the protein. Thus, a variant nucleic acid molecule can be produced by: one or more nucleotide substitutions, additions and/or deletions are introduced into the corresponding nucleic acid sequences disclosed herein such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleic acid sequences are also encompassed by the present disclosure.
Alternatively, variant nucleic acid sequences can be prepared by randomly introducing mutations along all or part of the coding sequence (e.g., by saturation mutagenesis), and the resulting mutants can be screened for the ability to confer activity to identify mutants that retain activity. Following mutagenesis, the encoded protein may be recombinantly expressed, and the activity of the protein may be determined using standard assay techniques.
The polynucleotides and fragments thereof of the present disclosure are optionally used as substrates for various recombinant and recursive (recursive) recombination reactions, in addition to standard cloning methods described, for example, by Ausubel, Berger, and Sambrook, i.e., to generate additional polypeptide homologs and fragments thereof having desired properties. Various such reactions are known. Methods for producing variants of any of the nucleic acids listed herein, which methods comprise recursive recombination of such polynucleotides with a second (or more) polynucleotide, thereby forming a library of variant polynucleotides, are also embodiments of the present disclosure, as are the libraries produced, cells comprising the libraries, and any recombinant polynucleotides produced by such methods. In addition, such methods optionally include selecting variant polynucleotides from such libraries based on activity, as where such recursive recombination is performed in vitro or in vivo.
Various diversity generation schemes, including nucleic acid recursive recombination schemes, are available and well described in the art. The programs can be used alone and/or in combination to generate one or more variants of a nucleic acid or collection of nucleic acids, as well as variants of the encoded protein. Individually or collectively, these procedures provide a robust and widely applicable way of generating diverse nucleic acids and collections of nucleic acids (including, for example, nucleic acid libraries) that can be used, for example, for the engineering or rapid evolution of nucleic acids, proteins, pathways, cells, and/or organisms with new and/or improved characteristics.
Although distinction and classification are made in the course of the following discussion for the sake of clarity, it should be understood that the techniques are generally not mutually exclusive. In practice, the various methods can be used alone or in combination, in parallel or in tandem, in order to obtain different sequence variants.
The result of any diversity generation procedure described herein can be the generation of one or more nucleic acids that can select or screen for nucleic acids having or conferring a desired property or nucleic acids encoding proteins having or conferring a desired property. Any nucleic acid produced may be selected for a desired activity or property, such as such activity at a desired pH, etc., after diversification by one or more methods herein or otherwise available to the skilled artisan. This may include identifying any activity that can be detected, for example, in an automated or automatable format, by any assay in the art. Various related (or even unrelated) characteristics may be evaluated in series or in parallel by the practitioner as appropriate.
The nucleotide sequences of the examples can also be used to isolate corresponding sequences from different sources. In this manner, such sequences can be identified using methods such as PCR, hybridization, and the like (based on their sequence homology to the sequences identified by the methods disclosed herein). The embodiments encompass sequences selected based on sequence identity to all sequences set forth herein or fragments thereof. Such sequences include sequences that are orthologs of the sequences. The term "ortholog" refers to a gene derived from a common ancestral gene and found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share substantial identity as defined elsewhere herein.
In the PCR method, oligonucleotide primers can be designed for use in a PCR reaction to amplify a corresponding DNA sequence 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 [ molecular cloning: a Laboratory Manual (2 nd edition, Cold Spring Harbor Laboratory Press, Plainview, New York), hereinafter "Sambrook". See also, edited by Innis et al, (1990) PCR Protocols: AGuide to Methods and Applications [ PCR protocol: methods and application guide ] (Academic Press, New York); edited by Innis and Gelfand, (1995) PCR Strategies [ PCR strategy ] (Academic Press, New York); and edited by Innis and Gelfand, (1999) PCR Methods Manual [ PCR Methods Manual ] (Academic Press, New York). Known PCR methods include, but are not limited to: methods using pair primers, nested primers, monospecific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like.
In hybridization methods, all or part of a nucleic acid sequence can be used to screen a cDNA or genomic library. Methods for constructing such cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook and Russell, (2001), supra. So-called hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group (e.g., 32P or any other detectable label, such as other radioisotopes, fluorescent compounds, enzymes, or enzyme cofactors). Probes for hybridization can be prepared by labeling synthetic oligonucleotides based on the nucleic acid sequences disclosed herein encoding known polypeptides. Degenerate primers may additionally be used, which are designed based on conserved nucleotides or amino acid residues in the nucleic acid sequence or the encoded amino acid sequence. Such probes typically comprise a region of nucleic acid sequence that hybridizes under stringent conditions to at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175, or 200 consecutive nucleic acids of a nucleic acid sequence encoding a polypeptide, or a fragment or variant thereof. Methods and stringent conditions for preparing probes for hybridization are generally known in the art and are disclosed in Sambrook and Russell, (2001), supra.
The use of the term "nucleotide construct" herein is not intended to limit the embodiments to nucleotide constructs comprising DNA. One of ordinary skill in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides composed of ribonucleotides, and combinations of ribonucleotides and deoxyribonucleotides, can also be used in the methods disclosed herein. The nucleotide constructs, nucleic acids and nucleotide sequences of the embodiments additionally encompass all complementary forms of such constructs, molecules and sequences. In addition, the nucleotide constructs, nucleotide molecules, and nucleotide sequences of the examples encompass all nucleotide constructs, molecules, and sequences that can be used in the methods of transforming plants of the examples, including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-loop structures, and the like.
Further embodiments relate to transformed organisms, such as organisms selected from the group consisting of: plant cells, bacteria, yeast, baculovirus, protozoa, nematodes and algae. The transformed organism comprises: the DNA molecule, expression cassette comprising the DNA molecule, or vector comprising the expression cassette of the embodiments can be stably incorporated into the genome of the transformed organism.
The sequences of the examples are provided in DNA constructs for expression in an organism of interest. The construct will include regulatory sequences operably linked to the 5 'and 3' of the sequences of the examples. As used herein, the term "operably linked" refers to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of a DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary, join two protein coding regions in the same reading frame. The construct may additionally contain at least one additional gene to be co-transformed into the organism. Alternatively, one or more additional genes may be provided on multiple DNA constructs.
Such DNA constructs are provided with multiple restriction sites for insertion of the polypeptide gene sequences of the present disclosure that will be under the transcriptional regulation of the regulatory regions. The DNA construct may additionally comprise a selectable marker gene.
In the 5 'to 3' direction of transcription, the DNA construct will typically comprise: a transcription and translation initiation region (i.e., a promoter), the DNA sequences of the examples, and a transcription and translation termination region (i.e., a termination region) that is functional in the organism used as the host. For the host organism and/or sequences of the embodiments, the transcriptional initiation region (i.e., promoter) may be native, analogous, exogenous, or heterologous. Furthermore, the promoter may be a natural sequence, or alternatively, a synthetic sequence. As used herein, the term "exogenous" means that the promoter is not found in the native organism into which it is introduced. Where a promoter is "exogenous" or "heterologous" to a sequence of an embodiment, it refers to a promoter that is not native or naturally occurring to the operably linked sequence of the embodiment. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcriptional initiation region that is heterologous to the coding sequence. When the promoter is a native (native or native) sequence, expression of the operably linked sequence is altered from wild-type expression, which results in an alteration of the phenotype.
In some embodiments, the DNA construct comprises a polynucleotide encoding the ZmMM1 polypeptide of the embodiments. In some embodiments, the DNA construct comprises a polynucleotide encoding a fusion protein comprising the ZmMM1 polypeptide of the embodiments.
In some embodiments, the DNA construct may further comprise a transcriptional enhancer sequence. As used herein, the term "enhancer" refers to a DNA sequence that can stimulate promoter activity, and can be an innate element or a heterologous element of a promoter inserted to enhance the level or tissue specificity of the promoter. Various enhancers are known in the art, including, for example, introns with Gene expression enhancing properties in plants (U.S. patent application publication No. 2009/0144863), ubiquitin introns (i.e., maize ubiquitin intron 1 (see, e.g., NCBI sequence S94464)), the omega enhancer or omega major enhancer (Gallie et al, (1989) Molecular Biology of RNA, Cech editing (List, New York) 237-. The above list of transcriptional enhancers is not meant to be limiting. Any suitable transcription enhancer may be used in the examples.
The termination region may be native to the transcriptional initiation region, native to the operably linked DNA sequence of interest, native to the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, sequence of interest, plant host, or any combination thereof).
Convenient termination regions may be obtained from the Ti plasmid of agrobacterium tumefaciens (a. tumefaciens), such as octopine synthase and nopaline synthase termination regions. See also Guerineau et al, (1991) mol. geh. genet [ molecular and general genetics ] 262: 141-144; proudfoot (1991) Cell [ Cell ] 64: 671-674; sanfacon et al, (1991) Genes Dev. [ Genes and development ] 5: 141-149; mogen et al, (1990) Plant Cell [ Plant Cell ] 2: 1261-; munroe et al, (1990) Gene [ Gene ] 91: 151-158; ballas et al, (1989) Nucleic Acids Res. [ Nucleic acid research ] 17: 7891-7903 and Joshi et al, (1987) Nucleic Acid Res [ Nucleic Acid research ] 15: 9627-9639.
Where appropriate, the nucleic acids may be optimized for increased expression in the host organism. Thus, where the host organism is a plant, the synthetic nucleic acid may be synthesized using plant-preferred codons to improve expression. For a discussion of the use of host preferences, see, e.g., Campbell and Gowri, (1990) Plant Physiol [ Plant physiology ] 92: 1-11. For example, although the Nucleic acid sequences of the examples may be expressed in both monocot and dicot plant species, the sequences may be modified to take into account the specific preferences and GC content preferences of monocot or dicot plants, as these preferences have shown differences (Murray et al (1989) Nucleic Acids Res. [ Nucleic Acids research ] 17: 477-. Thus, the plant preference of a particular amino acid can be derived from the known gene sequence of the plant.
Additional sequence modifications are known to enhance gene expression in cellular hosts. These include the elimination of the following sequences: sequences encoding pseudopolyadenylation signals, sequences encoding exon-intron splice site signals, sequences encoding transposon-like repeats, and other well-characterized sequences that may be detrimental to gene expression. The GC content of the sequence can be adjusted to the average level of a given cellular host, as calculated by reference to known genes expressed in the host cell. As used herein, the term "host cell" refers to a cell that contains a vector and supports replication and/or expression of an expression vector. The host cell may be a prokaryotic cell such as E.coli, or a eukaryotic cell such as a yeast, insect, amphibian, or mammalian cell, or a monocotyledonous or dicotyledonous plant cell. An example of a monocot host cell is a maize host cell. When possible, the sequence is modified to avoid the occurrence of predictable hairpin secondary mRNA structures.
In preparing the expression cassette, the various DNA segments can be manipulated to provide DNA sequences in the proper orientation and, where appropriate, in the proper reading frame. To this end, adapters (adapters) or linkers may be employed to ligate the DNA fragments, or other manipulations may be involved to provide convenient restriction sites, remove excess DNA, remove restriction sites, and the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, re-substitution (e.g. transitions and transversions) may be involved.
A number of promoters may be used in the practice of the embodiments. Promoters may be selected based on the desired result. The nucleic acid may be used in combination with constitutive, tissue-preferred, inducible or other promoters for expression in the host organism.
The methods of the embodiments involve introducing the polypeptide or polynucleotide into a plant. As used herein, "introducing" means presenting the polynucleotide or polypeptide to the plant in such a way that the sequence enters the interior of the plant cell. The methods of the embodiments do not depend on the particular method used to introduce one or more polynucleotides or one or more polypeptides into a plant, so long as the polynucleotides or polypeptides enter the interior of at least one cell of the plant. Methods for introducing one or more polynucleotides or one or more polypeptides into plants are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
As used herein, "stable transformation" means that a nucleotide construct introduced into a plant is integrated into the genome of the plant and is capable of being inherited by its progeny. As used herein, "transient transformation" means the introduction of a polynucleotide into the plant and not integrated into the genome of the plant, or the introduction of a polypeptide into a plant. As used herein, "plant" refers to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, and embryos and progeny thereof. Plant cells may be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, and pollen).
Transformation protocols, as well as protocols for introducing nucleotide sequences into plants, may vary depending on the type of plant or plant cell to be targeted for transformation (i.e., monocots or dicots). Suitable methods for introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al, (1986) Biotechniques [ Biotechnology ] 4: 320-, agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al, (1984) EMBO J [ J.Eur. Med. 3: 2717-Buffe 2722), and ballistic particle acceleration (see, e.g., U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes et al, (1995) Plant, Tissue, and Organ Culture: Fundamental Methods [ Plant cells, tissues and organs Culture: basic Methods ], Gamborg and Phillips editors (Springer-Verlag, Berlin [ Berlin Schlingge publication, Germany ]); and Mcbebec et al, (1988) Biotechnology [ Biotechnology ] 6: Buffe 926); and the Lecl transformation method (WO 00/28058). For potato transformation, see Tu et al, (1998) Plant Molecular Biology [ Plant Molecular Biology ] 37: 829-838 and Chong et al, (2000) Transgenic Research [ Transgenic Research ] 9: 71-78. Additional transformation methods can be found in the following references: weissinger et al, (1988) ann.rev.genet. [ yearbook of genetics ] 22: 421-477; sanford et al, (1987) Particulate Science and Technology [ microparticle Science and Technology ] 5: 27-37 (onions); christou et al, (1988) Plant Physiol [ Plant physiology ] 87: 671-674 (soybean); McCabe et al, (1988) Bio/Technology [ Bio/Technology ] 6: 923-; finer and McMullen, (1991) In Vitro Cell dev. biol. [ In Vitro Cell biology and developmental biology ] 27P: 175- & ltSUB & gt 182 & lt/SUB & gt (soybean); singh et al, (1998) the or. appl. genet [ theory and applied genetics ] 96: 319-324 (soybean); datta et al, (1990) Biotechnology [ Biotechnology ] 8: 736-740 (rice); klein et al, (1988) proc.natl.acad.sci.usa [ proceedings of the american academy of sciences ] 85: 4305-; klein et al, (1988) Biotechnology [ Biotechnology ] 6: 559-563 (maize); U.S. patent nos. 5,240,855; 5,322,783 and 5,324,646; klein et al, (1988) Plant Physiol [ Plant physiology ] 91: 440-444 (maize); fromm et al, (1990) Biotechnology [ Biotechnology ] 8: 833-; Hooykaas-Van Slogteren et al, (1984) Nature [ Nature ] (London) 311: 763 764; U.S. Pat. No. 5,736,369 (cereal); bytebier et al, (1987) Proc. Natl. Acad. Sci. USA [ Proc. Sci. USA ] 84: 5345-; de Wet et al, (1985) The Experimental management of Ovule Tissues [ Experimental procedures for Ovule organization ], Chapman et al, eds (Longman, Langmo, N.Y.), pp.197-; kaeppler et al, (1990) Plant Cell Reports 9: 415 and Kaeppler et al, (1992) the or. appl. Genet. [ theoretical and applied genetics ] 84: 560-566 (whisker-mediated transformation); d' Halluin et al, (1992) Plant Cell [ Plant Cell ] 4: 1495-1505 (electroporation); li et al, (1993) Plant Cell Reports, 12: 250-: 407-; osjoda et al, (1996) Nature Biotechnology [ Nature Biotechnology ] 14: 745-750 (maize via Agrobacterium tumefaciens).
In some embodiments, the polynucleotide composition may be introduced into the genome of the plant using genome editing techniques, or a previously introduced polynucleotide in the genome of the plant may be edited using genome editing techniques. For example, the identified polynucleotides can be introduced into the plant genome at desired locations by using double-strand break techniques (e.g., TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, etc.). For example, the CRISPR-Cas system can be used to introduce the identified polynucleotides into the genome at desired locations for the purpose of site-specific insertion. The desired location in the plant genome may be any target site required for insertion, such as a genomic region suitable for breeding, or may be a target site located in a genomic window with an existing trait of interest. The existing trait of interest may be an endogenous trait or a previously introduced trait.
In some embodiments, where the disease resistant ZmMM1 gene allele has been identified in the genome, the polynucleotide sequence may be altered or modified using genome editing techniques. Site-specific modifications that can be introduced into the desired ZmMM1 gene allele polynucleotide include modifications made using any method for introducing site-specific modifications, including, but not limited to, by using gene repair oligonucleotides (e.g., U.S. publication 2013/0019349), or by using double-strand break techniques such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. Such techniques can be used to modify previously introduced polynucleotides by insertion, deletion, or substitution of nucleotides within the introduced polynucleotide. Alternatively, additional nucleotide sequences may be added to the introduced polynucleotide using double strand break technology. Additional sequences that may be added include additional expression elements (e.g., enhancer sequences and promoter sequences). In another embodiment, genome editing techniques can be used to locate additional disease resistance proteins within the genome of a plant in proximity to the ZmMM1 polynucleotide composition to produce a molecular stack of disease resistance proteins.
"altered target site", "altered target sequence", "modified target site", and "modified target sequence" are used interchangeably herein and mean a target sequence as disclosed herein that comprises at least one alteration when compared to the unaltered target sequence. Such "changes" include, for example: (i) a substitution of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i) - (iii).
Examples
The following examples are provided to illustrate, but not to limit, the claimed subject matter. It is understood that the examples and embodiments described herein are for illustrative purposes only and that one skilled in the art will recognize that various reagents or parameters may be changed without departing from the spirit of the disclosure or the scope of the appended claims.
Example 1 ZmMM1 modulates a pathopoiesia phenotype in maize (loss-mimic phenotype)
C117 is from BC2F7A Near Isogenic Line (NIL) of a population derived from a single F1 cross between a plateau maize mexican (maize (Zea mays ssp. mexicana)) and a maize inbred Mo17, wherein Mo17 is the recurrent parent, exhibiting a patholike phenotype. By locating the F2 population from C117 and Mo17, a major QTL was identified on chromosome 7 that controls the pathopoiesia phenotype and was named qLMchr 7. qLMchr7 was finely mapped to a 5kb interval flanked by the markers M2(SEQ ID NOS: 27 and 28) and M3(SEQ ID NOS: 11 and 12) using a location-based cloning method (FIG. 1). There is only one gene annotated in this interval based on the maize mexican (teosinte) genomic sequence, which was named ZmMM1 (maize mexican (Zea mays Mexicana) type lesion 1). Further fine positioning will qLMchr7 is defined as the 1kb interval 950bp downstream of the CDS ZmMM1 (FIG. 1). The 1kb region is part of the ZmMM 13 'UTR, as determined by 3' RACE analysis. Within the 1kb qLMchr7 region, there were 20 SNPs and 7 indels shared between C117 and Mo17 (FIG. 1). Comparing the qLMchr7 sequence of C117 with the qLMchr7 sequences of 46 different maize inbred lines, it was found that the 30bp specific region in C117 (SEQ ID NO: 16; and corresponding to 24bp in Mo17, SEQ ID NO: 15) has two SNPs and one indel, where C117 has a unique haplotype (FIG. 1).
Because qLMchr7 is part of the 3' UTR of ZmMM1, qLMchr7 was tested for its function to see if it was dependent on ZmMM1, and if qLMchr7 is a regulatory element of ZmMM1 expression. Although the transcriptional levels of ZmMM1 in Mo17 and C117 leaves were similar, the level of ZmMM1 protein as determined by Western blotting using anti-ZmMM 1 antibody was found to contain the C117 qLMchr7 allele (qLMchr 7)c117) Is higher than in the Mo17 qLMchr7 allele (qLMchr 7)Mo17). This confirms qLMchr7c117Ratio qLMchr7Mo17Resulting in higher levels of ZmMM1 protein.
Transient overexpression of C117 or Mo17 ZmMM1 CDS with the 35S promoter in Nicottana benthamiana (Nicottana benthamiana) resulted in cell death. When a 1kb qLMchr7 fragment (SEQ ID NOS: 13 and 14) was inserted between the ZmMM1 CDS and the terminator sequence, the vector carries qLMchr7C1The ZmMM1 construct of 17 still caused cell death in Nicotiana benthamiana (N.benthamiana), but with qLMchr7 aloneMo17The construct of (a) induces a weak cell death phenotype. Although both alleles of qLMchr7 significantly reduced the transcript and protein levels of ZmMM1 compared to the construct without the qLMchr7 fragment, but with qLMchr7c117And qLMchr7Mo17There was no difference in the level of ZmMM1 transcript between constructs. However, with qLMchr7Mo17In contrast, with qLMchr7c117The constructs of (a) yielded higher levels of ZmMM1 protein, consistent with expression results in maize. In addition, qLMchr7 was replaced by the corresponding 30bp in C117Mo17Of (3) (resulting in qLMchr 7)Mo17-m) Increase ZmMM1 protein levels and lead to strong cell death in Nicotiana benthamiana (N.benthamiana)Phenotype. In contrast, qLMchr7 was replaced by the corresponding 24bp in Mo17C117Of (3) (resulting in qLMchr 7)C117-m) Reduce the ZmMM1 protein level and result in a weak cell death phenotype in nicotiana benthamiana (n. Thus, it can be concluded that qLMchr7 regulates the expression of ZmMM1 at the protein level, while higher ZmMM1 protein levels are associated with a patholike phenotype in C117.
Loss-of-function ZmMM1 mutant alleles were identified from the B73 EMS mutagenized population. This mutant allele (ZmMM1-1) has an inadvertent mutation in the second exon of ZmMM1, which introduces a premature stop codon. Overexpression of zmmm1-1 did not result in Nicotiana benthamiana cell death. The zmmm1-1 mutant was combined with a plasmid bearing qLMchr7c117Allelic plants were crossed and a pair of NILs was identified in the subsequent F3 population. Both NILs have the same qLMchr7C117Allele (1kb fragment). However, one had wild type ZmMM1 from C117 (ZmMM1-qLMchr 7)C117) And the other has a mutated zmmm1-1 allele (zmmm1-1-qLMchr 7)C117)。ZmMM1-qLMchr7C117Plants showed clear lesion-like phenotype, but zmmm1-1-qLMchr7C117The plant did not. The observations confirmed that ZmMM1 is responsible for the pathopoiesia phenotype in C117.
Example 2 Forward Regulation of ZmMM1 on northern leaf blight (NLB, also known as northern leaf Spot) and Gray leaf Spot (GLS, also known as northern leaf Spot) Known as gray leaf spot) and Southern Corn Rust (SCR)
The zmmm1-1 homozygous mutant plants were crossed with B73 plants to produce F1 plants and a population of F2. F2 plants with homozygous ZmMM1-1 allele were significantly more susceptible to NLB and GLS than F2 plants with wild-type ZmMM1, as determined by lesion length in field infected plants. Zmmm1-1 mutant F2 plants were also more susceptible to SCR than wild-type F2 plants after inoculation of conidia of puccinia mays in the greenhouse as determined by visual comparison of fungal biomass accumulation (qRT-PCR amplification of actin mRNA of puccinia mays) and the amount of endospores. Thus, it was concluded that knockout of ZmMM1 increases maize susceptibility to NLB, GLS and SCR.
Two pairs of carriers qLMchr7 were evaluated in the fieldC117Or qLMchr7Mo17Resistance of the allelic NIL to NLB, GLS and SCR. The disease phenotype is rated 1-9, "1" is the most resistant, and "9" is the most susceptible. Having qLMchr7C117The NIL ratio of the alleles has qLMchr7Mo17The NILs of the alleles are more resistant to NLB, GLS and SCR. The results indicate that plants with the zea mexicana (teosinte) ZmMM1 allele are more resistant to multiple pathogens than plants with the maize Mo17 ZmMM1 allele.
Example 3 identification of ZmMM1 protein target genes
ZmMM1(CDS sequence-SEQ ID NO: 9; genomic sequence-SEQ ID NO: 5) encodes a transcription factor containing a MYB DNA binding domain (SEQ ID NO: 3). The determination of transcriptional activity in protoplasts shows that ZmMM1 significantly inhibits the expression of reporter gene GUS when fused with the DNA Binding Domain (BD) of GAL4, which contains four GAL4 DNA binding sites in the promoter, indicating that ZmMM1 is a transcriptional repressor. DNA affinity purification sequencing (DAP-seq) was performed and four candidate ZmMM1 target genes (ZmMT1, ZmMT2, ZmMT3 and ZmMT4) were identified. The ChIP-qPCR assay confirmed direct binding of the ZmMM1 protein to the promoter regions of the four target genes. Finally, transient expression of ZmMT3(SEQ ID NO: 18) in Nicotiana benthamiana (N.benthamiana) inhibited ZmMM 1-induced cell death. Since ZmMM1 positively regulates disease resistance and negatively regulates expression of its target genes, down-regulation in ZmMT1(SEQ ID NOS: 20 and 21), ZmMT2(SEQ ID NO: 23), ZmMT3 or ZmMT4(SEQ ID NO: 25) enhances resistance to a variety of pathogens. ZmMT1(SEQ ID NOS: 20 and 21), ZmMT2(SEQ ID NO: 23) and ZmMT3(SEQ ID NO: 18) are long non-coding RNAs (lncRNA), while ZmMT4(SEQ ID NO: 25) encodes a polypeptide.
Sequence listing
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Lys Trp Met Ser Thr Ala Gln Leu Trp Thr Gly Asp Ser Gly Arg Glu
115 120 125
Asp Ala Glu Ser Glu Lys Gln Asp Lys Gly Arg Cys Ser Pro Glu Ala
130 135 140
Arg Ser Arg Gly Ala Leu Leu Pro Phe Lys Ala Asp Val Gly Ser Gly
145 150 155 160
Ala Pro Ala Phe Ala Pro Leu Phe Leu Arg Thr Asp Asp Lys Ala Ala
165 170 175
Ala Ala Arg Val Gly Val Pro Asp Leu Ser Ser Leu Leu Ser Pro Pro
180 185 190
Ala Thr Met Pro Pro Ala Asp Ala Gly Ala Glu Glu Ser Arg Arg Gln
195 200 205
Val Val Gly Phe Ala Gln Ala Ala Ala Arg Ala Ala Ala Met Ala Pro
210 215 220
Ser Ala Pro Ala Leu Gly Leu Gln Ser Gln Gln Gln Gln Gln Gln Gln
225 230 235 240
Gln Gln Ala Arg Lys Ala Arg Arg Cys Trp Ser Thr Glu Leu His Arg
245 250 255
Lys Phe Val Ala Ala Leu Asp Gln Leu Gly Gly Pro Gln Val Ala Thr
260 265 270
Pro Lys Gln Ile Arg Glu Leu Met Lys Val Asp Gly Leu Thr Asn Asp
275 280 285
Glu Val Lys Ser His Leu Gln Lys Tyr Arg Leu His Asn Arg Arg Ala
290 295 300
Pro Gly Ser Gly Val Val Arg Gln Pro Ile Val Leu Val Gly Gly Leu
305 310 315 320
Trp Ile Pro Gln Glu Gln Gly Ser Pro Gln Ser Gly Ser Pro His Gly
325 330 335
Pro Leu His His Leu Ser Thr Ser Val Ala Ala Val Ser Ser Ala Ala
340 345 350
Thr Ala Ser Cys Glu Glu Glu Asp Gly Arg Ser Glu Ser Tyr Gly Trp
355 360 365
Lys
<210> 2
<211> 365
<212> PRT
<213> maize
<400> 2
Met Gly Leu Asp Val Met Glu Ile Gly Met Gly Ala Asp Leu Ser Leu
1 5 10 15
Asp Leu Arg His Phe Ala Ser Lys Ala Val Arg Gln Ser Lys Asp Asp
20 25 30
Thr Pro Ala Pro Asp Met Asp Ala Cys Ile Arg Arg Leu Glu Glu Glu
35 40 45
Arg Gly Lys Ile Glu Met Phe Lys Arg Asp Leu Pro Leu Cys Ala Arg
50 55 60
Leu Leu Ala Asp Val Ile Asp Val Met Lys Glu Glu Ala Gly Lys Lys
65 70 75 80
Lys Thr Thr Thr Arg Arg Ser Asp Arg Arg Leu Ala Ser Ala Ala Ala
85 90 95
Asp Glu Glu Glu Glu Glu Glu Asp Gly Ala Thr Ala Asp Lys Ser Lys
100 105 110
Trp Met Ser Thr Ala Gln Leu Trp Thr Gly Asp Ser Gly Arg Glu Asp
115 120 125
Ala Glu Ser Glu Lys Gln Asp Lys Gly Trp Cys Ser Pro Glu Ala Arg
130 135 140
Ser Arg Gly Ala Leu Leu Pro Phe Lys Ala Glu Val Gly Ser Gly Ala
145 150 155 160
Pro Ala Phe Ala Pro Leu Cys Leu Arg Thr Asp Asp Lys Ala Ala Ala
165 170 175
Ala Arg Val Gly Val Pro Asp Leu Ser Ser Leu Leu Ser Ser Pro Ala
180 185 190
Thr Met Pro Pro Ala Asp Ala Gly Ala Glu Glu Ser Arg Arg Gln Val
195 200 205
Val Gly Phe Ala Gln Ala Ala Ala Arg Ala Ala Ala Met Ala Pro Ser
210 215 220
Ala Pro Ala Leu Gly Leu Gln Ser Gln Gln Gln Gln Gln Gln Ala Arg
225 230 235 240
Lys Ala Arg Arg Cys Trp Ser Thr Glu Leu His Arg Lys Phe Val Ala
245 250 255
Ala Leu Asp Gln Leu Gly Gly Pro Gln Val Ala Thr Pro Lys Gln Ile
260 265 270
Arg Glu Leu Met Lys Val Asp Gly Leu Thr Asn Asp Glu Val Lys Ser
275 280 285
His Leu Gln Lys Tyr Arg Leu His Asn Arg Arg Ala Pro Gly Ser Gly
290 295 300
Val Val Arg Gln Pro Ile Val Leu Val Gly Gly Leu Trp Ile Pro Gln
305 310 315 320
Glu Gln Gly Ser Pro Gln Ser Gly Ser Pro His Gly Pro Leu His His
325 330 335
Leu Ser Thr Ser Val Ala Ala Val Ser Ser Ala Ala Thr Ala Ser Cys
340 345 350
Glu Glu Glu Asp Gly Arg Ser Glu Ser Tyr Gly Trp Gln
355 360 365
<210> 3
<211> 367
<212> PRT
<213> maize
<400> 3
Met Gly Leu Asp Val Met Glu Ile Gly Met Gly Ala Asp Leu Ser Leu
1 5 10 15
Asp Leu Arg His Phe Ala Ser Lys Ala Val Arg Gln Ser Lys Asp Asp
20 25 30
Thr Pro Ala Pro Asp Met Asp Ala Cys Ile Arg Arg Leu Glu Glu Glu
35 40 45
Arg Gly Lys Ile Glu Met Phe Lys Arg Asp Leu Pro Leu Cys Ala Arg
50 55 60
Leu Leu Ala Asp Val Ile Asp Val Met Lys Glu Glu Ala Gly Lys Lys
65 70 75 80
Thr Thr Thr Thr Arg Arg Ser Asp Arg Arg Leu Ala Ser Ala Ala Ala
85 90 95
Asp Glu Glu Glu Glu Glu Glu Asp Gly Ala Thr Ala Asp Lys Ser Lys
100 105 110
Trp Met Ser Thr Ala Gln Leu Trp Thr Gly Asp Ser Gly Arg Glu Asp
115 120 125
Ala Glu Ser Glu Lys Gln Asp Lys Gly Arg Cys Ser Pro Glu Ala Arg
130 135 140
Ser Arg Gly Ala Leu Leu Arg Phe Lys Ala Asp Val Gly Ser Gly Ala
145 150 155 160
Pro Ala Phe Ala Pro Leu Cys Leu Arg Thr Asp Asp Lys Ala Ala Ala
165 170 175
Ala Arg Val Gly Val Pro Asp Leu Ser Ser Leu Leu Ser Pro Pro Ala
180 185 190
Thr Met Pro Pro Ala Asp Ala Gly Ala Glu Glu Ser Arg Arg Gln Val
195 200 205
Val Gly Phe Ala Gln Ala Ala Ala Arg Ala Ala Ala Met Ala Pro Ser
210 215 220
Ala His Ala Leu Gly His Gln Ser Gln Ser Gln Gln Gln Gln Gln Gln
225 230 235 240
Ala Arg Lys Ala Arg Arg Cys Trp Ser Thr Glu Leu His Arg Lys Phe
245 250 255
Val Ala Ala Leu Asp Gln Leu Gly Gly Pro Gln Val Ala Thr Pro Lys
260 265 270
Gln Ile Arg Glu Leu Met Lys Val Asp Gly Leu Thr Asn Asp Glu Val
275 280 285
Lys Ser His Leu Gln Lys Tyr Arg Leu His Asn Arg Arg Ala Pro Gly
290 295 300
Ser Gly Val Val Arg Gln Pro Ile Val Leu Val Gly Gly Leu Trp Ile
305 310 315 320
Pro Gln Glu Gln Gly Ser Pro Gln Ser Gly Ser Pro His Gly Pro Leu
325 330 335
His His Leu Ser Thr Ser Val Ala Ala Val Ser Ser Ala Ala Thr Ala
340 345 350
Ser Cys Glu Glu Glu Asp Gly Arg Ser Glu Ser Tyr Gly Trp Lys
355 360 365
<210> 4
<211> 5006
<212> DNA
<213> maize
<400> 4
cgttcacgcg atttttcaag tgaaagcgag accgaaaacc agcaatgggc tgtaggaatg 60
cattaggata tttttaccaa atattagcca aacgtttttg ttattatgca taatagtagg 120
catagcacta aatttacaat agactacaaa tataggggct ttattacaaa atattctttc 180
ggacgtgaaa actcgtcatt gttgattcgg agattctacc actccatctc cagttttttc 240
cctcgcctgc tcagctcccc tataaatgga gctcgccttc cgcggcctcc ctccgttccc 300
atccgccgcc cgcgcacttc ttccttcggg cacacaggac accaccgtcg acggattcat 360
cgcgacgatg gggctcgacg tcatggagat cgggatgggc gccgatttga gcctggatct 420
gaggcacttc gcctccaagg ccgtgaggca gagcaaggac gacacgccgg cgccggacat 480
ggacgcatgc atccgccgcc tcgaggagga gcggggtaag atcgagatgt tcaagcggga 540
cctcccgctc tgcgcgcgcc tcctcgccga cggtgagcgc acctacctct tctcctctct 600
ctctgtctct ctctcttttt atttttccca cctgtgattc atttgggata ccttttgctt 660
ctttccattt tggggagcgg ttttttttac gcggcgatgc ggtggcgtgt gcgcagtaat 720
tgatgtcatg aaggaggagg cggggaagaa gaagacgacg acaaggagga gtgaccgcag 780
gctggcgtct gcggcagctg atgaggagga ggaggaggag gacggcgcca ccgcggacaa 840
gagcaagtgg atgagcacgg cgcagctctg gacgggcgat tccgggcggg aggacgcgga 900
atcagaggta cggcacgatt cgatcgctgg tgcagctgct tgaatgctca gtcagcacag 960
gatctggagg gtgctgtcgg gtgctcgatt cgtcggcagg cctaaaagtt tggagctttg 1020
cgatcgcaga agcaagacaa ggggtggtgc tcgccggagg ccaggtcccg cggcgctctc 1080
ttaccgttca aggctgaagt gggctctggc gcgccggcgt tcgcgccgct ctgcttgaga 1140
acggacgaca aggctgcggc tgcgcgcgtc ggggtgccgg atctgtcgtc cttgctgtcg 1200
tcgccggcga ccatgcctcc tgcggacgcc ggcgccgagg agagccgtcg ccaggttgtg 1260
ggatttgcgc aagctgcggc cagggcggct gccatggcgc cgtctgcccc tgcgcttggg 1320
ctccagtcgc agcagcagca gcagcaggca aggaaggctc ggcgttgctg gtcgacggag 1380
ctgcatcgca agttcgtcgc cgccttggat cagctcggtg gcccccaagg tgagccttgc 1440
cttgttcttc ggatgccagt tcaccagaat ctcttgccag ttttgagcca ccaacacgtt 1500
caatcttacc tagttgctag ctgccttcca tattagaatc ataaaattgg gatcaatgag 1560
tctatgccat gactgcagtt gccacgccga agcaaatcag ggagctgatg aaggtggatg 1620
ggctgacaaa cgacgaagtg aaaagccatc ttcaggttag cgatccagca gcagctcact 1680
ccccttgcca ttccattcat ccatctcatc tcaggaagtc acgagtatct gttgttgtga 1740
tggttgctga aatggattct ccgatttcga tgtctcttca gaaataccgg ctgcacaacc 1800
ggagggcgcc tggatccggc gtggtgcgcc agccgatcgt gctcgtggga gggctgtgga 1860
ttccccagga gcaaggcagc cctcagtctg gatctcccca cggccccctc caccacctgt 1920
ccacctcggt ggccgccgtc tcgtccgccg ccaccgccag ctgcgaggag gaagacggcc 1980
ggtccgagag ctatggctgg caatgatgtc tggctgctgc tgctgctgca ccaccaatgt 2040
gtgttcactg ttcagagagg ggaggtttct tggcatggtg gggatcgcca tgggccatgg 2100
cggaggccac cagttgcagc ttcaggaatc gggaggggaa ttgagtgtag tgtagctgtc 2160
tgtacacata catacataca tacagtgaga tgggatgaga tgagagcggg ccttgagcgc 2220
tcgagatcag aactgatggt gcttcgtcgt cgggtttgta catcccaaag agaaagagat 2280
actagctaca gttttgcggc ttgttaatcc atgctctggg ggcagagcta cagttttcgc 2340
cccgagagag ttcacccata cccgttgttg tcgattagac gattaccatc ttcgccttct 2400
tgttgccgtt gaacaaaatg ttgcttccgc tgttcgtctc ggaacgaaca gtccggttga 2460
aaagttgaat cgttgcagga gtacatgcta ctcaggctgt aatgtggttg gtaagggtgt 2520
ttgaatgaac tagacctaat agttagtgac taaaattagt tggatacatc taaacatcct 2580
ataatcctat agtttaacta ttagatattt gttatctcgc taattttata agtaattttt 2640
agccaactaa ctattagttc taatgcattc gaacgctcac tctgcagctt tccagtcgcg 2700
tatcgttcag gtctatctaa ctgaaagagc agcaaagaag aaagagatct caataagaaa 2760
agaacccgat tccaccattg aacaaccaac caagagggtt gctcctgctc ctctgctgtt 2820
caccatcatc aacagcacgt aaaaaaaatc tctagctctc tactttaccg tctaccagta 2880
gtgtttggat aaagcaacag aagccggctt ctcttctttt tttgcagaga taatatagat 2940
attcagaaga agaaaaaaga attatctggg cctaactgaa actgagctga cggagcacga 3000
gcacggaagc catgcttgtt gtatacataa cataagccgg ggggaggata tgctcgaggc 3060
attctcttct tcctcctccg tcagtcactg gctcggtcca ttcgttagcg tctcaccagt 3120
ccttgatcag cattgttaat actactagct cgctgctgag tgctgacaat gcgaaacagt 3180
ttcttggcag gattccaact cgagctcgcc gtcgctgtcg ctgtcgctgg accgtaggaa 3240
cgtgccggtc ccctccctgc atggcgggcc aaagagccac ccagatcaga ggacggatcc 3300
ccgtgaaatc ccccctcttg ttctttaatt actcgcaggc ggaggaaagg cggcagtgca 3360
cagcgacaga gagacgaaga ctttggaatc gtccttgggt ggatggatgg acggacgaac 3420
gaggcggggc cgcgagctct gaatattcgc cgccgtcgat gcatcggcgg cctgcctgtc 3480
gctgtcgacg gagagggtgg tactggtgtg cgcaaccgga caacgcaatg ttcaggcctg 3540
aagaatcgga atcggaatat tatattccgt ctggtgtggt tgtcactttc ttttctttgt 3600
gtgtgttttt ttgttgtttg ttgttgttgt gtagataaat actatgggga agaatggagg 3660
gggatatgag gatatcctcg ttgattctgc ttgagaaact agggcgtatt atatgataca 3720
tacatttgga attctcactc tcggccgacc ccgccggcga cctcagcccg acgtggatat 3780
ataaaaaaaa agatgattaa agctttgtaa gataagacta gtctgcactt tctagtagat 3840
ttagaccata ttttgaaacg tctgaaacta ataattaaca gataaaacta gctaagagag 3900
aacgaatcag ctaatagatt agctaattgc tggttacatc tctcaaatag ctattagtta 3960
ttagttaatt taatctagct aaaatcaaat acaacaactt actctttatg tacaaaatta 4020
aaatttgttt tagtttttta ttggattcat ataataattt gtgtttatgt ttttttatat 4080
gtttctaaat ttattatata aaaactaaga gataaaatga ataataattt ttggacggaa 4140
agaatattag ctccctacag ttttgggaca ggtcgcaact tgtctgacta gttaagtttc 4200
tatagcgcgg tagtagtcta gtagaagata gtactctctt tgtttctttt tagttattat 4260
tggataattt aattttgtaa tattcagcga caactaaaac gaaacgtaag agagggtaga 4320
taactttgga gacttgagtc gtcgtgaatg ggatgggact tgtcggagcc tcggcgcagc 4380
gtattatttg ttgacagggc cgtgagagcc tggtccacat tttgttggcc catttaggtg 4440
agctgtctac agattgggcc gagcaagtaa gggtatagga agccgaaatg tgcccattta 4500
accaatcatc acggtttgag tcgacattcc acgattctgc aaccacagta ctttatttat 4560
ttggttgaaa acacaaggtt aattaatact aacagtagcg acaatgatga tgctccttca 4620
cgcttccttg ccatatcata aaaaaacagt aaaaaggtaa aagaaaaagg ttaatgccta 4680
cctatagctt ttagcttgca gcgcgccctc tctctcttct ctctgtatat atgccgtgat 4740
cgccggcaca catcgcggcg tgtttgcatt ccgatcggcg gccgcgaaaa aggaaaaata 4800
aagaagtgaa aaatagagga aagcagaaag aataaaagag ggctaaaaga aaaaggcatg 4860
tcgccgtatg ctggcgcctt gatagtcagg ggctcagggc gtcaggcaga catgcttgta 4920
gtagttagta tatagagtcc ggccgacacg gttcagcggc cacatgcatg cagcgacagg 4980
ctaatcaaag cccacaacag agaccg 5006
<210> 5
<211> 5256
<212> DNA
<213> maize
<400> 5
cgttcacgcg atttttcaag tgaaagccaa atattagcca aacgtttttg ttattatgca 60
taatagtagg catagcacta aatttacaat agactacaaa tataggggct ttcgtacaaa 120
ataatctttc ggacgtgaaa actcgtcatt gttgattcgg agattctacc actccatctc 180
cagttttttt ttccctcgcc tgctcggctc ccctataaat ggagctcacc ttccgcggcc 240
tccctccgtt cccatccgcc gcccgcgcac ttcttccttc gggcacacag gacaccaccg 300
tcgacggatt catcgcgacg atggggctcg acgtcatgga gatcgggatg ggcgccgatt 360
tgagcctgga tctgaggcac ttcgcctcca aggccgtgag gcagagcaag gacgacacgc 420
cggcgccgga catggacgca tgcatccgcc gcctcgagga ggagcggggt aagatcgaga 480
tgttcaagcg ggacctcccg ctctgcgcgc gcctcctcgc cgacggtgag cgcacctacc 540
tcttctctct ctgtctctct ctctttttta tttttcccac ctgtgattca tttgggatac 600
cttctgcttc tttccatttt ggggagcggg tttttttatg cggcgatgtg gtggcgtgtg 660
cgcagtaatt gatgtcatga aggaggaggc ggggaagaag acgacgacca cgaggaggag 720
tgatcgcagg ctggcgtctg cggcagctga tgaggaggag gaggaggagg acggcgccac 780
cgcggacaag agcaagtgga tgagcacggc gcagctctgg acgggcgatt ccgggcggga 840
ggacgcggaa tcagaggtac ggcacgattc gatcgctggt gcagctgctt gaatgcccag 900
tcagcacagg atctgggggg tgctgtcggg tgctcgattc gtcggcaggc ctaaaagttt 960
tggagctttg cgatcgcaga agcaagacaa ggggcggtgc tcgccggagg ccaggtcccg 1020
cggcgctctc ttacggttca aggctgatgt gggctctggc gcgccggcgt tcgcgccgct 1080
ctgcttgaga acggacgaca aggctgcggc tgcgcgcgtc ggggtgccgg atctgtcgtc 1140
cttgctgtcg ccgccggcga ccatgcctcc tgcggacgcc ggcgccgagg agagccgtcg 1200
ccaggttgtg ggatttgcgc aagctgcggc cagggcggct gccatggcgc cgtctgccca 1260
tgcgcttggg caccagtcgc agtcgcagca gcagcagcag caggcaagga aggctcggcg 1320
ttgctggtcg acggagctgc atcgcaagtt cgtcgccgcc ttggatcagc tcggtggccc 1380
ccaaggtgag ccttgccttg ttcttcggat gccagttcac cagaatttct tgccagtttt 1440
gggccaccaa cacacacgtt caatcttacc tagttgctag ctgccttcca tattatatta 1500
gaaacactga gttcattcat gctacgccat gcctgcagtt gccacgccga agcaaatcag 1560
ggagctgatg aaggtggatg ggctgacaaa cgacgaagtg aaaagccatc ttcaggttag 1620
cgatccagca gcagctcact ccccttgaca ttccattcat ccatctcatc tcaggaagtc 1680
acgaatatct gttgttgtga tggttgctga aatggattct ctgatttcga tgtttgttca 1740
gaaataccgg ctgcacaacc gcagggcgcc tggatccggc gtggtgcgcc agccgatcgt 1800
gctcgtggga gggctgtgga ttccccagga gcaaggcagc cctcagtctg gatctcccca 1860
cggccctctc caccacttgt ccacctcggt ggccgccgtc tcgtccgccg ccaccgccag 1920
ctgcgaggag gaagacggcc ggtccgagag ctatggctgg aaatgatgaa gaggctgctg 1980
ctgctgctgc accaccaatg tgtgttcact gtttagagag gggaggtttc ttggcatggt 2040
ggggatcgcc atgggccatg gcggaggcca ccagttgcag cttcaggaat cgggagggga 2100
attgagtgta gtgtagctgt ctgtacacat acatacatac atacattgag atgggatgag 2160
atgagagcgg gccttgagcg ctcgagatca gaactgatgg tgcttcgtcg tcgggtttgt 2220
acatcccaaa gagaaagaga aagagatact agctacagtt ttgcggcttg ctaatccatg 2280
cctgggggca gagctacagt tttcgccccg agagagttca cccatcaccc atacccgttg 2340
ttgtcgatta ccatcttcgc cttcttgttg ccgttgaaca aaatgttgct ttcgctgttc 2400
gtctcggaac gaacagtccg gttgaaaagt tgaatcgttg caggagtaca tgctactgag 2460
tctgtaatgt ggttggtaag ggtgtttgaa tgaactagac ctaatagtta gtgactaaaa 2520
ttagaatcat atagtttaac tattagatat ttgttatctc gctaatttta taagtaattt 2580
ttagccaact aactattagt tcgaatgcat tcgaacactc actctgcagc tttccagtcg 2640
cgtatcgtta aggtctatct aactgaaaga gcagcaaaga tgaaagagat ctcaataaga 2700
aaagaacccg attccaccat tgaacaacca accaagaggg ttgctcctgc tcctctgctg 2760
ttcaccatca tcaacagcac gtaaaaaaaa aatctctagc tctctactgt accgtctacc 2820
agtagtgttt ggataaagca acagacgccg gtttctcttc ttttttacag agataatata 2880
gatattcaga agaagaagaa aaaggaataa ttatctgggc ctaactgaaa ctgagctgac 2940
ggagcacgag cacggaagcc atgcttgttg tatacataac ataagccggg gggaggatat 3000
gctcgaggca ttctcttctt cctcatccgt cagtcactgg ctcggtccat tcgttagcgt 3060
ctcaccagtc cttgatcagc attgttaata ctactagctc gctgctgagt gctgactatg 3120
cgaaacagtt tcttggcagg attccaacaa ctcgagctcg ccgtcgccgt cgctgtcgct 3180
ggaccgtacg aacgtgccgg tcccctccct gcatggcgga ccaaagagcc acccagatca 3240
ggacggatcc ccgtgaaatc ccccctcttg ttctttaatt actcgcaggc ggaggaaagg 3300
cggcagtgca cagcgacaga gagacgaaga ctttggaatc gtccttgggt gcatggatgg 3360
acggacggac gaacgagagg gggggccgcg agctctgaat attcgccgcc gtcgatgcat 3420
cggcggcctg cctgtcgctc tcgacggaga gggtggtact ggtgtgcgca accggacaac 3480
gcaatgttca ggccagaaga atcggaatcg gaatatcata ttccgtctgg tgtggtggtc 3540
actttctttt ctttgtgtgt gtgttttttt gttgttgttg tgtagataaa tactatgggg 3600
aagaatggag gggatatgag gatatcctcg ttgattctgc ttgagaaact agggcgtatt 3660
atatgataca tttggaattc tcactctcgg ctgggccgac cccgccggcg acctcagccc 3720
gacgtggata tatataaaaa gatgattaaa gctttgtaag ataagactag tctgcacttt 3780
ctagtagatt tagaccatat tttcaaacgt ctaaaactaa taatgaacag ataaaactag 3840
ctaagagaga acgaatcagc taatagatta gctaattgtt agttacattt ctcaaatagc 3900
tattagttgt tagttaattt aatctagcta aaatcaacta caacaactta ctctttatgt 3960
acaaaattaa aatttgtttt agttttttat tggattcata taataatttg tgtttatgtt 4020
tttttatatg tttctaaatt tattatataa aaactaagag ataaaatgaa taataatttt 4080
tggacggaaa gaatattagc tccctacagt tttgggacag gtcgcaactt gtctgactag 4140
ttaagtttct atagcgcggt agtagtctag tagaagatag tactctcttt gtttcttttt 4200
agttattatt ggataattta attttgtaat attcagcgac aactaaaacg aaacgtaaga 4260
gagggtagat aactttggag acttgagtcg tcgtgaatgg gatgggactt gtcggagcct 4320
cggcgcagcg tattatttgt tgacagggcc gtgagagcct ggtccacatt ttgttggccc 4380
atttaggtga gctgtctaca gattgggccg agcaagtaag ggtataggaa gccgaaatgt 4440
gcccatttaa ccaatcatca cggtttgagt cgacattcca cgattctgca accacagtac 4500
tttatttatt tggttgaaaa cacaaggtta attaatacta acagtagcga caatgatgat 4560
gctccttcac gcttccttgc catatcataa aaaacagtaa aaaggtaaaa gaaaaaggtt 4620
aatgcctacc tatagcttgc agcgcgccct ctctctcttc tctctgtata tatgccgtga 4680
tcgccggcac acatcgcggc gtgtttccat tccgatcggc ggcggcgaaa aagggaaaaa 4740
taaagaagta aaaaatagag gaaagcagaa agaaaaaaaa aagggttaaa agaaaaggca 4800
tgtcgccgta tgctggcgcc ttgatagtca ggcagacatg ctttgcagta gtagtagtat 4860
atagggtgtg tttggtttga cttttgactc tggcttttac cccctaaaag ctaaaagcca 4920
aaccaaaggg ctggatttag gaagcagctt tttctaaaag ccgactttct tgcagtgcaa 4980
aactgaaagc acctctagac ctgcttttag ctgcttttag atggaactgt gaaaatatat 5040
atggaaaaac atttagcgac ttttagtggt ttccaccaaa cactttttag ctttttaaca 5100
gctcgcagcc cacagcagct tttctcacag ctcacagccc acagcagctt ttttcacagc 5160
cacagtccaa ccaaacagac catagagtcc ggccgacacg gttcagcggc cacatgcatg 5220
cagcgacagt ctaatcaaag cccacaacag agaccg 5256
<210> 6
<211> 2084
<212> DNA
<213> maize
<400> 6
gttcccatcc gccgcccgcg cacttcttcc ttcgggcaca caggacacca ccgtcgacgg 60
attcatcgcg acgatggggc tcgacgtcat ggagatcggg atgggcgccg atttgagcct 120
ggatctgagg cacttcgcct ccaaggccgt gaggcagagc aaggacgaca cgccggcgcc 180
ggacatggac gcatgcatcc gccgcctcga ggaggagcgg ggtaagatcg agatgttcaa 240
gcgggacctc ccgctctgcg cgcgcctcct cgccgacggt gagcgcacct acctcttctc 300
ctctcttctg tctctctctt ttttattttt cccacctgtg attcatttgg gataccttct 360
gcttctttcc attttgggga gcggtttttt ttatgcggcg atgcggtggc gtgtgcgcag 420
taattgatgt catgaaggag gaggcgggga agaagaagac gacgacgagg aggaggagtg 480
atcgcaggct ggcgtctgcg gcagctgatg atgaggagga ggaggcggac ggcgccaccg 540
cggacaagag caagtggatg agcacggcgc agctctggac gggcgattcc gggcgggagg 600
acgcggaatc agaggtacgg cacgattcga tcgctggtgc agctgcttga atgctcagtc 660
agcacaggat ctgtgggggt gctgtcgggt gctcgattcg tcggtgggcc taaaagtttt 720
ggagctttgc gatcgcagaa gcaagacaag gggcggtgct cgccggaggc caggtcccgc 780
ggcgctctct taccgttcaa ggctgatgtg ggctctggcg cgccggcgtt cgcgccgctc 840
ttcttgagaa cggacgacaa ggctgcggct gcgcgcgtcg gggtgccgga tctgtcgtcc 900
ttgctgtcgc cgccggcgac catgcctcct gcggacgccg gcgccgagga gagccgtcgc 960
caggttgtgg gatttgcgca agctgcggcc agggcggctg ccatggcgcc gtctgcccct 1020
gcgcttgggc tccagtcgca gcagcagcag cagcagcagc agcaggcaag gaaggctcgg 1080
cgttgctggt cgacggagct gcatcgcaag ttcgtcgccg ccttggatca gctcggtggc 1140
ccccaaggtg agccttgcct tgttcttcgg atgccagttc accagaattt cttgccagtt 1200
ttgggccacc aacacacacg tccaatctta cctagttgct agctgccttc catattatat 1260
tagaaagaaa cattgagttc attcatgcta cgccatgcct gcagttgcca cgccgaagca 1320
aatcagggag ctgatgaagg tggatgggct gacaaacgac gaagtgaaaa gccatcttca 1380
ggttagcgat ccagcagcag ctcagtccac ttggcattcc attcatccat ctcaggaagt 1440
cacgaatatc tgttgttttg atggttgctg aaatggattc tctaattccg atgtttattc 1500
agaaataccg gctgcacaac cgcagggcgc ctggatccgg cgtggtgcgc cagccgatcg 1560
tgctcgtggg agggctgtgg attccccagg agcaaggcag ccctcagtct ggatctcccc 1620
acggccccct ccaccacctg tccacctcgg tggccgccgt ctcgtccgcc gccaccgcca 1680
gctgcgagga ggaagacggc cggtccgaga gctatggctg gaaatgatga agaggctgct 1740
gctgctgctg ctgcgccacc aatgtgtgtt cactgtttag agaggggagg gaggtttctt 1800
ggcatggtgg ggatcgccat gggccatggc ggaggccacc agttgcagct tcaggaatcg 1860
ggaggggaat tgagtgtagt gtagctgtct gtacacatac atacatacat acagtgagat 1920
gggatgagat gagagcgggc cttgagcgct cgagatcaga actgatggtg cttcgtcgtc 1980
gggtttgtac atcccaaaga gaaagagata ctagctacag ttttgcggct tgttaatcca 2040
tgctctgggg gcagagctac agttttcgcc ccgagagagt tcac 2084
<210> 7
<211> 1540
<212> DNA
<213> maize
<400> 7
gttcccatcc gccgcccgcg cacttcttcc ttcgggcaca caggacacca ccgtcgacgg 60
attcatcgcg acgatggggc tcgacgtcat ggagatcggg atgggcgccg atttgagcct 120
ggatctgagg cacttcgcct ccaaggccgt gaggcagagc aaggacgaca cgccggcgcc 180
ggacatggac gcatgcatcc gccgcctcga ggaggagcgg ggtaagatcg agatgttcaa 240
gcgggacctc ccgctctgcg cgcgcctcct cgccgacgta attgatgtca tgaaggagga 300
ggcggggaag aagaagacga cgacgaggag gaggagtgat cgcaggctgg cgtctgcggc 360
agctgatgat gaggaggagg aggcggacgg cgccaccgcg gacaagagca agtggatgag 420
cacggcgcag ctctggacgg gcgattccgg gcgggaggac gcggaatcag agaagcaaga 480
caaggggcgg tgctcgccgg aggccaggtc ccgcggcgct ctcttaccgt tcaaggctga 540
tgtgggctct ggcgcgccgg cgttcgcgcc gctcttcttg agaacggacg acaaggctgc 600
ggctgcgcgc gtcggggtgc cggatctgtc gtccttgctg tcgccgccgg cgaccatgcc 660
tcctgcggac gccggcgccg aggagagccg tcgccaggtt gtgggatttg cgcaagctgc 720
ggccagggcg gctgccatgg cgccgtctgc ccctgcgctt gggctccagt cgcagcagca 780
gcagcagcag cagcagcagg caaggaaggc tcggcgttgc tggtcgacgg agctgcatcg 840
caagttcgtc gccgccttgg atcagctcgg tggcccccaa gttgccacgc cgaagcaaat 900
cagggagctg atgaaggtgg atgggctgac aaacgacgaa gtgaaaagcc atcttcagaa 960
ataccggctg cacaaccgca gggcgcctgg atccggcgtg gtgcgccagc cgatcgtgct 1020
cgtgggaggg ctgtggattc cccaggagca aggcagccct cagtctggat ctccccacgg 1080
ccccctccac cacctgtcca cctcggtggc cgccgtctcg tccgccgcca ccgccagctg 1140
cgaggaggaa gacggccggt ccgagagcta tggctggaaa tgatgaagag gctgctgctg 1200
ctgctgctgc gccaccaatg tgtgttcact gtttagagag gggagggagg tttcttggca 1260
tggtggggat cgccatgggc catggcggag gccaccagtt gcagcttcag gaatcgggag 1320
gggaattgag tgtagtgtag ctgtctgtac acatacatac atacatacag tgagatggga 1380
tgagatgaga gcgggccttg agcgctcgag atcagaactg atggtgcttc gtcgtcgggt 1440
ttgtacatcc caaagagaaa gagatactag ctacagtttt gcggcttgtt aatccatgct 1500
ctgggggcag agctacagtt ttcgccccga gagagttcac 1540
<210> 8
<211> 1098
<212> DNA
<213> maize
<400> 8
atggggctcg acgtcatgga gatcgggatg ggcgccgatt tgagcctgga tctgaggcac 60
ttcgcctcca aggccgtgag gcagagcaag gacgacacgc cggcgccgga catggacgca 120
tgcatccgcc gcctcgagga ggagcggggt aagatcgaga tgttcaagcg ggacctcccg 180
ctctgcgcgc gcctcctcgc cgacgtaatt gatgtcatga aggaggaggc ggggaagaag 240
aagacgacga caaggaggag tgaccgcagg ctggcgtctg cggcagctga tgaggaggag 300
gaggaggagg acggcgccac cgcggacaag agcaagtgga tgagcacggc gcagctctgg 360
acgggcgatt ccgggcggga ggacgcggaa tcagagaagc aagacaaggg gtggtgctcg 420
ccggaggcca ggtcccgcgg cgctctctta ccgttcaagg ctgaagtggg ctctggcgcg 480
ccggcgttcg cgccgctctg cttgagaacg gacgacaagg ctgcggctgc gcgcgtcggg 540
gtgccggatc tgtcgtcctt gctgtcgtcg ccggcgacca tgcctcctgc ggacgccggc 600
gccgaggaga gccgtcgcca ggttgtggga tttgcgcaag ctgcggccag ggcggctgcc 660
atggcgccgt ctgcccctgc gcttgggctc cagtcgcagc agcagcagca gcaggcaagg 720
aaggctcggc gttgctggtc gacggagctg catcgcaagt tcgtcgccgc cttggatcag 780
ctcggtggcc cccaagttgc cacgccgaag caaatcaggg agctgatgaa ggtggatggg 840
ctgacaaacg acgaagtgaa aagccatctt cagaaatacc ggctgcacaa ccggagggcg 900
cctggatccg gcgtggtgcg ccagccgatc gtgctcgtgg gagggctgtg gattccccag 960
gagcaaggca gccctcagtc tggatctccc cacggccccc tccaccacct gtccacctcg 1020
gtggccgccg tctcgtccgc cgccaccgcc agctgcgagg aggaagacgg ccggtccgag 1080
agctatggct ggcaatga 1098
<210> 9
<211> 1104
<212> DNA
<213> maize
<400> 9
atggggctcg acgtcatgga gatcgggatg ggcgccgatt tgagcctgga tctgaggcac 60
ttcgcctcca aggccgtgag gcagagcaag gacgacacgc cggcgccgga catggacgca 120
tgcatccgcc gcctcgagga ggagcggggt aagatcgaga tgttcaagcg ggacctcccg 180
ctctgcgcgc gcctcctcgc cgacgtaatt gatgtcatga aggaggaggc ggggaagaag 240
acgacgacca cgaggaggag tgatcgcagg ctggcgtctg cggcagctga tgaggaggag 300
gaggaggagg acggcgccac cgcggacaag agcaagtgga tgagcacggc gcagctctgg 360
acgggcgatt ccgggcggga ggacgcggaa tcagagaagc aagacaaggg gcggtgctcg 420
ccggaggcca ggtcccgcgg cgctctctta cggttcaagg ctgatgtggg ctctggcgcg 480
ccggcgttcg cgccgctctg cttgagaacg gacgacaagg ctgcggctgc gcgcgtcggg 540
gtgccggatc tgtcgtcctt gctgtcgccg ccggcgacca tgcctcctgc ggacgccggc 600
gccgaggaga gccgtcgcca ggttgtggga tttgcgcaag ctgcggccag ggcggctgcc 660
atggcgccgt ctgcccatgc gcttgggcac cagtcgcagt cgcagcagca gcagcagcag 720
gcaaggaagg ctcggcgttg ctggtcgacg gagctgcatc gcaagttcgt cgccgccttg 780
gatcagctcg gtggccccca agttgccacg ccgaagcaaa tcagggagct gatgaaggtg 840
gatgggctga caaacgacga agtgaaaagc catcttcaga aataccggct gcacaaccgc 900
agggcgcctg gatccggcgt ggtgcgccag ccgatcgtgc tcgtgggagg gctgtggatt 960
ccccaggagc aaggcagccc tcagtctgga tctccccacg gccctctcca ccacttgtcc 1020
acctcggtgg ccgccgtctc gtccgccgcc accgccagct gcgaggagga agacggccgg 1080
tccgagagct atggctggaa atga 1104
<210> 10
<211> 349
<212> DNA
<213> maize
<400> 10
cagacatgct ttgcagtagt agtagtatat agggtgtgtt tggtttgact tttgactctg 60
gcttttaccc cctaaaagct aaaagccaaa ccaaagggct ggatttagga agcagctttt 120
tctaaaagcc gactttcttg cagtgcaaaa ctgaaagcac ctctagacct gcttttagct 180
gcttttagat ggaactgtga aaatatatat ggaaaaacat ttagcgactt ttagtggttt 240
ccaccaaaca ctttttagct ttttaacagc tcgcagccca cagcagcttt tctcacagct 300
cacagcccac agcagctttt ttcacagcca cagtccaacc aaacagacc 349
<210> 11
<211> 20
<212> DNA
<213> maize
<400> 11
cggtctctgt tgtgggcttt 20
<210> 12
<211> 20
<212> DNA
<213> maize
<400> 12
atgctggcgc cttgatagtc 20
<210> 13
<211> 1048
<212> DNA
<213> maize
<400> 13
aattatctgg gcctaactga aactgagctg acggagcacg agcacggaag ccatgcttgt 60
tgtatacata acataagccg gggggaggat atgctcgagg cattctcttc ttcctcctcc 120
gtcagtcact ggctcggtcc attcgttagc gtctcaccag tccttgatca gcattgttaa 180
tactactagc tcgctgctga gtgctgacaa tgcgaaacag tttcttggca ggattccaac 240
tcgagctcgc cgtcgctgtc gctgtcgctg gaccgtagga acgtgccggt cccctccctg 300
catggcgggc caaagagcca cccagatcag aggacggatc cccgtgaaat cccccctctt 360
gttctttaat tactcgcagg cggaggaaag gcggcagtgc acagcgacag agagacgaag 420
actttggaat cgtccttggg tggatggatg gacggacgaa cgaggcgggg ccgcgagctc 480
tgaatattcg ccgccgtcga tgcatcggcg gcctgcctgt cgctgtcgac ggagagggtg 540
gtactggtgt gcgcaaccgg acaacgcaat gttcaggcct gaagaatcgg aatcggaata 600
ttatattccg tctggtgtgg ttgtcacttt cttttctttg tgtgtgtttt tttgttgttt 660
gttgttgttg tgtagataaa tactatgggg aagaatggag ggggatatga ggatatcctc 720
gttgattctg cttgagaaac tagggcgtat tatatgatac atacatttgg aattctcact 780
ctcggccgac cccgccggcg acctcagccc gacgtggata tataaaaaaa aagatgatta 840
aagctttgta agataagact agtctgcact ttctagtaga tttagaccat attttgaaac 900
gtctgaaact aataattaac agataaaact agctaagaga gaacgaatca gctaatagat 960
tagctaattg ctggttacat ctctcaaata gctattagtt attagttaat ttaatctagc 1020
taaaatcaaa tacaacaact tactcttt 1048
<210> 14
<211> 1048
<212> DNA
<213> maize
<400> 14
aattatctgg gcctaactga aactgagctg acggagcacg agcacggaag ccatgcttgt 60
tgtatacata acataagccg gggggaggat atgctcgagg cattctcttc ttcctcatcc 120
gtcagtcact ggctcggtcc attcgttagc gtctcaccag tccttgatca gcattgttaa 180
tactactagc tcgctgctga gtgctgacta tgcgaaacag tttcttggca ggattccaac 240
aactcgagct cgccgtcgcc gtcgctgtcg ctggaccgta cgaacgtgcc ggtcccctcc 300
ctgcatggcg gaccaaagag ccacccagat caggacggat ccccgtgaaa tcccccctct 360
tgttctttaa ttactcgcag gcggaggaaa ggcggcagtg cacagcgaca gagagacgaa 420
gactttggaa tcgtccttgg gtgcatggat ggacggacgg acgaacgaga gggggggccg 480
cgagctctga atattcgccg ccgtcgatgc atcggcggcc tgcctgtcgc tctcgacgga 540
gagggtggta ctggtgtgcg caaccggaca acgcaatgtt caggccagaa gaatcggaat 600
cggaatatca tattccgtct ggtgtggtgg tcactttctt ttctttgtgt gtgtgttttt 660
ttgttgttgt tgtgtagata aatactatgg ggaagaatgg aggggatatg aggatatcct 720
cgttgattct gcttgagaaa ctagggcgta ttatatgata catttggaat tctcactctc 780
ggctgggccg accccgccgg cgacctcagc ccgacgtgga tatatataaa aagatgatta 840
aagctttgta agataagact agtctgcact ttctagtaga tttagaccat attttcaaac 900
gtctaaaact aataatgaac agataaaact agctaagaga gaacgaatca gctaatagat 960
tagctaattg ttagttacat ttctcaaata gctattagtt gttagttaat ttaatctagc 1020
taaaatcaac tacaacaact tactcttt 1048
<210> 15
<211> 24
<212> DNA
<213> maize
<400> 15
gatggatgga cggacgaacg aggc 24
<210> 16
<211> 30
<212> DNA
<213> maize
<400> 16
catggatgga cggacggacg aacgagaggg 30
<210> 17
<211> 10105
<212> DNA
<213> maize
<400> 17
cggtgtgtac aaagggcagg gacgtagtca acgcgagctg atgactcgcg cttactaggc 60
attcctcgtt gaagaccaac aattgcaatg atctatcccc atcacgatga aatttcccaa 120
gattacccgg gcctgtcggc caaggctata tactcgttgg atacatcagt gtagcgcgcg 180
tgccgcccag aacatctaag ggcatcacag acctgttatt gcctcaaact tccgtggcct 240
aaacggccat agtccctcta agaagctaac tacggaggga tggctccgca tagctagtta 300
gcaggctgag gtctcgttcg ttaacggaat taaccagaca aatcgctcca ccaactaaga 360
acggccatgc accaccaccc atagaatcaa gaaagagctc tcagtctgtc aatccttgct 420
atgtctggac ctggtaagtt tccccgtgtt gagtcaaatt aagccgcagg ctccacgcct 480
ggtggtgccc ttccgtcaat tcctttaagt ttcagccttg cgaccatact ccccccggaa 540
cccaaagact ttgatttctc ataaggtgcc agcggggtcc tattagtaac acccgctgat 600
ccctggtcgg catcgtttat ggttgagact aggacggtat ctgatcgtct tcgagccccc 660
aactttcgtt cttgattaat gaaaacatcc ttggcaaatg ctttcgcagt tgttcgtctt 720
tcataaatcc aagaatttca cctctgacta tgaaatacga atgcccccga ctgtccctat 780
taatcattac tccgatcccg aaggccaaca caataggacc ggaatcctat gatgttatcc 840
catgctaatg tatccagagc gatggcttgc tttgagcact ctaatttctt caaagtaacg 900
gcgccggagg cacgacccgg ccagttaagg ccaggagcgc atcgccggca gaagggtcga 960
gccggtcggt tctcgccgtg aggcggaccg gccggcccgg cccaaggtcc aactacgagc 1020
tttttaactg caacaactta aatatacgct attggagctg gaattaccgc ggctgctggc 1080
accagacttg ccctccaatg gatcctcgtt aagggattta gattgtactc attccaatta 1140
ccagacacta acgcgcccgg tattgttatt tattgtcact acctccccgt gtcaggattg 1200
ggtaatttgc gcgcctgctg ccttccttgg atgtggtagc cgtttctcag gctccctctc 1260
cggaatcgaa ccctaattct ccgtcacccg tcaccaccat ggtaggcccc tatcctacca 1320
tcgaaagttg atagggcaga aatttgaatg atgcgtcgcc ggcacgaagg ccgtgcgatc 1380
cgtcaagtta tcatgaatca tcggatcggc gggcagagcc cgcgtcagcc ttttatctaa 1440
taaatgcgcc cctcccggaa gtcggggttt gttgcacgta ttagctctag aattactacg 1500
gttatccgag tagcacgtac catcaaacaa actataactg atttaatgag ccattcgcag 1560
tttcacagtt cgaattagtt catacttgca catgcatggc ttaatctttg agacaagcat 1620
atgactactg gcaggatcaa ccaggtagca cgtcctcgca gacgggccag cgccggcctc 1680
cgcgcggagg cgtcgtgccg ggctggcagt cgttcattcg ggcggaccga ttcttgggcg 1740
cgtgacgcca acgcgtctcc ggccttcagc gtgagccaca tccgagacca aaagcgccag 1800
cgaggtgtcc tcggtgccgc cggccatagg ccgacggcgg cacgaggcaa acgccgcgag 1860
cgctctcgag ccgacgagcc gcaccccggg gggtgagctc gacgaaggca acgtgtatcg 1920
agcacggctt cccgtgggac gggtagcagc acgcaagcac ttctcaacgc agcaggcata 1980
ggatgcccgc acgagcgatg ggacacaggc gccgggagtc ggccgcacgg cagcgggggt 2040
cctccaagca gtcacgggtc caagacaact catgcgcctg cgtagccgct acggtcgagc 2100
catccaaagc atccctccgc gctgggcgcg gcgggtctgc ttgcgaggac ggcgaccgaa 2160
ggtccaccga gcgcgggaga aacggaaaac gcatcgagca acgggccatc ccacggtgca 2220
gccactcgtc cagggcgtct ggccggcggt agccagccat agccggtcgt ggctgcgtca 2280
cggccgaacc acggccggcc aggcagccaa cagcgccagc cggagctggg cgcggtaggg 2340
tgccgaccgg ccacggctag gccgcgaggg ggtgcggggc tcggccgagg agacctggag 2400
gagacgctgg aaacgctatg gtttcagcag cgtttcgccc gggtttcggc tgcacgagtt 2460
ccctacccct actatacctg aggggcatac cccctcccag gacttcgggg agttctgcct 2520
tcagaaaacc agggcatttt cccagtaccc cacgaaaccc atctaagatg gctggacaca 2580
gcgtttttgc tcagaatcag gggtttcgct agcgtgaccc gttttccctc acgggtgcac 2640
ccgaacttcc acgtctcacg cggggcgacc acgggagggt cccgtgccct tccacgcgcc 2700
cgttttcgcg gccgtggccg aaaatccgtt tttggcccgt tcgccatggc gaacccctcg 2760
ttttcagcca aaacgcaagg ccgaacagcc ctgccgcccg ttgccttgcg tctcctcccg 2820
ttttccctcc gttccaccgt gcctttcaac cgagacctac gtagcaggct cggtgtcttt 2880
ccacgcgctt ggacttagcc cgttttcgcg gccgtggctg aaccgctgat ttcggccagc 2940
gcgccatggc gaacacctcg ttttcggccc agacgcaagg ccgaacagcc ctgccgcccg 3000
tcgccgcgcg cctcctcccg ttttccctcc gttccacctt gaccttcact ccagacatgc 3060
gctctaggtt cggaataaat atttgatttt accatatctc gcttgggcaa tgtttcatat 3120
gttatgcatg tagtatggac atggctctca aacctacaga cctagcatct tttttttaaa 3180
aaatagcgtc agcatacata ggccctctaa cccttgtctg tgtaaatccg tgagcatcct 3240
agctaggtgc ctaggtgagg aggtaccatg gttaatctat aactactgat agcagaaatc 3300
aataactctc aacaactact ccctccctcc gttctctatc ttaatatcat aaaggtttat 3360
cctaaatcaa tattttaaaa ctttaatagt taataataaa tattcataaa tattaactaa 3420
ataaaaataa aactattaca tttattatta aaacatctac tatacaatat ataaaaataa 3480
ttatttttat agagattgtt agtcaaagtt ttaaaatttt gacttagaac aaaccttcgt 3540
gaccttaaga tagggaacag atgaagtatt atatatgctg ccatgcccca tcgaaacaaa 3600
tcaagtgata ccatcaatat agctgcaaca gctgaaaact tgaaagggaa atgtgccttt 3660
gggccatttc taagtatttt tggtgattta gtgtctaaca caagtgccta agtgttgatc 3720
tatgcaaagt ggtggacaaa gtgtaaatca agtcaaaagg tatgtttcta gacttagtac 3780
attgttttat ggactgatgt attgtgtcta agtgctggaa acaggagaaa tcaaattgga 3840
aaagagatgt ctttgttcag ccaaagtctg ggtgcaccgg actgtccggt ggtgcaccag 3900
acagtgtccg gtgcgccagg cagactcagg cgaacttgct gctctcggga agtaattaac 3960
ggcgtacggc taaaattcac cggactgtcc ggtgagccaa cggtcggcca ggccaacagt 4020
cggccgggcc aacggtcggc cgcgcgatcc gcgcaggaca cgtggccgag ccaacggcta 4080
gtaggggcac cggactgtcc ggtgtggacc agacagtgtc cgatgcgcca acggctccaa 4140
ggctgccaac ggtcggcttc gccaaataag gaaggaaatc cgcaccggac tgtgcggtgg 4200
tgcaccggac agtccggtgc gccaggcgac agaaggcaag aattgccttc ccagattgct 4260
ctcaacggct cctagctgcc ttggggctat aaaagggacc cctaggcgca tggaggaaag 4320
aaccaagcat cctttgagca ttgttgatca ctcacactcc gttcttgcgc acttgttcga 4380
cattcttagt gatttgagct ccgttctagt gtgaaacttg tgatagtctc ttgagctcaa 4440
gtctgggtct tgtgtgtgcg tatttgctgt gatctttgtg tcttgtgtga gttgctcatc 4500
cctcccttac ttcgtgcttc tttgtgaaca tcaaagtgta agggcgagag gctccaagtt 4560
gtggagattc ctcgcgaacg ggatatagaa aagaaaagca aaacaccatg gtattcaagt 4620
gggtctttgg accgcttgag aggggttgat tgcaaccctc gtccgttggg acgccacaac 4680
gtggaagtag gcaagtgttg tacttggccg aaccacagga taaaccactg tgtctatttg 4740
tgttgattct gttgtggtta ttgtgtttcg ctaagactct tctctagcca cttggcatta 4800
ctgtgctaac gctgagagca cctagagggg ggggtgaata ggtgatcctg taaaacttaa 4860
acttatagcc acagaaactt ggttaatcgt tagcacaata attgccaagt ggctagagag 4920
gagtcaaaac acaataacca caagaaatca atcacagaga tgacacggtg gttatcccgt 4980
ggttcggcca agtacaaaac ttgcctactc cacgttgtgg cgtcccaacg gacgagagtt 5040
gcactcaact cctctcaagt gatccaatga tcaacttgaa taccacggtg ttcttcttta 5100
ctttgatctt ttcccgtttg cgaggaatct ccacaacttg gagtctctcg cccttacaat 5160
tgaatttcac aaagaagcac ggagtaaggg agggaagcaa cacacacaaa tccacagcaa 5220
tatgcgcaca cacacggcca agaatcgagc tcaaaagact atctcaaaat tctcactaga 5280
acggagctcg aattactgag aatgacaaat gaatgcgcaa agactgagtg tggatgatca 5340
agaatgctct aaggttgctt ggataactcc tccatgcgcc taggggtccc ttttatagcc 5400
ccaaggcagc taggagccgt tgagagcaaa tctggaagac caatcttgcc ttctgtcgtc 5460
gggtgcaccg gacaatccgg tgcacaccgg acactgtccg gtgcccgatt tctttcctta 5520
aacggcgcag tcgaccgttg ccgaccgttg cagatctggg agccgttggc gcaccggaca 5580
tgtccggtgc acaccggaca gtccggtgcc cccttccgac cgttggccag gccacgtgtc 5640
gcgcgcagat tccgcggccg accgttggct cggccgaccg ttggctcacc ggacagtccg 5700
gtgcacaccg gacagtccgg tgaattatag ccgtacgcca tcggcgaatt cccgagagcg 5760
gccacttcgc gccgtgtcag cctggcgcac cggacactgt ccggtgcacc accggacagt 5820
ccggtgtgct agaccgagct gagtcttggc tgtacacagc caagtctttg cacctttctt 5880
cttttctttt tctttctgtt tctaacactt agacaagtat attagtacac aaaaccaatg 5940
tactaaggct tagaaacata cctttactca tgatttgcac tttgttcatc catgggcata 6000
gattcacatt taagcacttg tgttggcact caatcaccaa aatactttag aaatggccca 6060
aaggcacatt tccctttcaa tctccccctt tttggtgatt tatgccaaca caacataaag 6120
caactagaac aagtgcaata tcacttcaaa taaaaataat tttgagtttt attcgatctt 6180
ggcatatatg gatcatcctt tgccaccact tggtttgttt ttgcaaatca aacacaaaat 6240
cctatctcta agtcaaatcc acttgtagag acacaaagag aggttttcca aagaaaattg 6300
attcaagatt ccaaaaactc cccctttttc ccataatcaa cacttctccc acaagagacc 6360
aacttttgac aaaagagaca atgcaagagt tttgaccaca caaaagctct aatctactat 6420
tttcaaaatt ctcaagtggt agctgatcca tttattgctt tggcctttat tttctccccc 6480
tttggcatca agcaccaaaa cgggattaat cttggcccta gaaccccatt gcctcaccaa 6540
aatcttcaac gaagaacaaa tagcaataag agttcatgag gtgaacttgg aataagttac 6600
cctctcatcg gagtgcagtg gaagtctttc atggtccaag tccacctttt ccctttcaat 6660
tctccttcga gactaaataa cgcaaactca agcatatggt tagtctcaaa agggtcaagt 6720
tgtaacacaa ctcccccaaa atatgtgcat cacttacaca aggacttgtg aggtccaggg 6780
aatgtttgta caacttgagc accacaataa gcaacaaaaa tgcagaatga acatgatcaa 6840
aggcataaac acatgtatgc tacaattcaa tccaagttcc gcgaatctaa gacatttagc 6900
tcactacgca gcctgcaaaa ggtcttctca tctagaggct tggtaaagat atcggctagc 6960
tggttctcgg tgctaacatg aaacacttcg atatctccct tttgctggtg gtctctcaaa 7020
aagtgatgcc ggatgtctat gtgctttgtg cggctgtgct caacaggatt ctccgccatg 7080
cggatagcac tctcattatc acataggagt gggactttgc tcagattgta gccaaagtcc 7140
cggagggttt gcctcatcca aagtagttgc gcgcaacact gtcctgcggc aacatactcg 7200
gcctcagcgg tggatagggc aacggaggtt tgtttcttag atttccacga caccagggac 7260
cttcctaaga attggcacgt ccctggtgta ctctttctat cgaccttaca tccagcatag 7320
tcggaatctg aatatccaat caagtcaaag gtagacccct ttggatacta gagcccgaag 7380
caaggcgtag ccaccaaata tctaagaatt cgcttcaccg ccactaagtg acactcctta 7440
ggatcggatt gaaatctagc acacatgcat acgctaagca taatatccgg tctactagca 7500
cataaataaa gtaaagaccc tatcattgac cggtatgctt tttgatcaac ggacttacct 7560
cctttgttga ggtcggtgtg tccgtcggtc cccatcggag tctttgcggg cttggcgtcc 7620
ttcatcccaa accgctttag cagatcttgc gtgtacttcg tttgggagat gaaggtgccg 7680
tccttgagtt gcttcacttg gaacccaagg aagtagttca actcgcccat cattgacatc 7740
tcgaatttct gcgtcatcac cctgctaaac tcttcacaag acttttggtt agtagaacca 7800
aatattatgt catcgacata aatttggcac acaaacaaat caccatcaca tgtctttgta 7860
aaaagagttg gatcggcttt cccaaccttg aaagcattaa caattagaaa gtctctaagg 7920
cattcatacc atgctcttgg ggcttgctta agtccataga gcgccttaga gagcttacac 7980
acgtggtcgg ggtaccgttc atcctcgaag ccagggggtt gctccacgta cacctcctcc 8040
ttgattggcc cattgaggaa agcgctcttc acatccattt ggaacaacct gaaagaatgg 8100
tgagcggcat atgctagcaa gatacgaatg gactctagcc tagccacagg agcaaaagtc 8160
tcctcaaagt ccaaacctgc gacttgggca taaccttttg ccacaagtcg agccttattc 8220
cttgtcacca ctccgtgctc gtcttgtttg ttgcggaaca cccacttggt tcccacaaca 8280
ttttgcttag gacgaggcac cagcgtccaa acttcattgc gcttgaagtt gttgagttcc 8340
tcctgcatgg ccaacaccca gtccggatct agcaaggcct cttctatcct gaaaggctca 8400
atagaagaga caaaggagta atgctcacaa aaattaacta atcgagatcg agtagttact 8460
cccttgctaa tgtcacccag aatttggttg acgggatgat ccctttgaat catcgctcga 8520
acttgggttg gaggtgccgg ttccgcttct tcctccatca catgatcatc ttgtgctccc 8580
ccttgatcac acgcctcctg ttgatgaacc tgttcatcgt cttgagttgg gggatgcacc 8640
attgttgagg aagaaggttg atctcgttca tcttgttcct gtggccgaac ttctccaatc 8700
gccatggttc gaatagcggc cgtcggaaca tcttcttcat ctacatcatc aagatcaaca 8760
atttgctctc ttggagagcc attagtctca tcaaatacaa cgtcgctaga gacttcaacc 8820
aaacccgatg atttgttgaa gactctatac gcctttgtat ttgagtcata acctaacaaa 8880
aacccttcta cagctttggg agcaaactta gaatttctac ccttctttac tagaatgtag 8940
cacttgctcc caaatacacg aaagtaagat acattgggtt tgttaccggt tagtagctca 9000
tacgacgtct tcttgaggag gcgatgaagg tagaccctgt tgatggcgtg gcaagccgtg 9060
ttcacggctt cagtccaaaa gcactcgggg gtcttgaact ctcctagcat cgtcctcgcc 9120
atatcgatga gcgtcctgtt cttcctttct accacaccgt tttgctgtgg tgtgtaggga 9180
gcggagaact tgtgcttgat cccttcctct tcaaggaact cctccacttg aaggttcttg 9240
aactcggacc cgttgtcgct ccttatcttc tttactttga gctcaaactc attttgagct 9300
ctcctgagga agcgcttgag ggtcccttgt gtttcagact tatcctgcaa aaagaacacc 9360
caagtgaagc gggaaaagtc atcaacaata actagacctt acttacttcc tcctatgctc 9420
agataggcga cgggtccgaa gaggtccata tgtagcagct ccagtggtct tgaggtggtc 9480
atcacattct tgctgtgatg tgttcctccc acttgtttac ctgcttgaca cgctgcacaa 9540
ggtctatctt tttcgaattg aacgttagtc aaacctatta cgtgttctcc ctttagaagc 9600
ttgtgaaggt tcttcatccc cacatgtgct aagcggcgat gccacagcca gcccatgcta 9660
gtcttagcta ttaagcatgc atctagaccg gcctcttctt ttgcaaaatc aactaaataa 9720
agtttgtcgt ctaatacacc cttaaaagct actgaaccat cacttcttct aaagacagac 9780
acatctacat ttgtaaatag acagttatat cccatattgc ataattgact aaccgatagc 9840
aaattatatc ctagagactc tactaaaaac acattagaga tagagtgctc attagagatt 9900
gcaattttac ctaacccttt tatcttgcct tgattcccat caccgaatat gattgaatct 9960
tgggaatcct tattcttgac gtaggaggtg aacatcttct tctcccccgt catatggttt 10020
gtgcatccgc tgtcaataat ccagcttgaa cccccggatg cataaacctg caaggcaaat 10080
ttaggcttgg gttttaggta cccaa 10105
<210> 18
<211> 1652
<212> DNA
<213> maize
<400> 18
cggtgtgtac aaagggcagg gacgtagtca acgcgagctg atgactcgcg cttactaggc 60
attcctcgtt gaagaccaac aattgcaatg atctatcccc atcacgatga aatttcccaa 120
gattacccgg gcctgtcggc caaggctata tactcgttgg atacatcagt gtagcgcgcg 180
tgccgcccag aacatctaag ggcatcacag acctgttatt gcctcaaact tccgtggcct 240
aaacggccat agtccctcta agaagctaac tacggaggga tggctccgca tagctagtta 300
gcaggctgag gtctcgttcg ttaacggaat taaccagaca aatcgctcca ccaactaaga 360
acggccatgc accaccaccc atagaatcaa gaaagagctc tcagtctgtc aatccttgct 420
atgtctggac ctggtaagtt tccccgtgtt gagtcaaatt aagccgcagg ctccacgcct 480
ggtggtgccc ttccgtcaat tcctttaagt ttcagccttg cgaccatact ccccccggaa 540
cccaaagact ttgatttctc ataaggtgcc agcggggtcc tattagtaac acccgctgat 600
ccctggtcgg catcgtttat ggttgagact aggacggtat ctgatcgtct tcgagccccc 660
aactttcgtt cttgattaat gaaaacatcc ttggcaaatg ctttcgcagt tgttcgtctt 720
tcataaatcc aagaatttca cctctgacta tgaaatacga atgcccccga ctgtccctat 780
taatcattac tccgatcccg aaggccaaca caataggacc ggaatcctat gatgttatcc 840
catgctaatg tatccagagc gatggcttgc tttgagcact ctaatttctt caaagtaacg 900
gcgccggagg cacgacccgg ccagttaagg ccaggagcgc atcgccggca gaagggtcga 960
gccggtcggt tctcgccgtg aggcggaccg gccggcccgg cccaaggtcc aactacgagc 1020
tttttaactg caacaactta aatatacgct attggagctg gaattaccgc ggctgctggc 1080
accagacttg ccctccaatg gatcctcgtt aagggattta gattgtactc attccaatta 1140
ccagacacta acgcgcccgg tattgttatt tattgtcact acctccccgt gtcaggattg 1200
ggtaatttgc gcgcctgctg ccttccttgg atgtggtagc cgtttctcag gctccctctc 1260
cggaatcgaa ccctaattct ccgtcacccg tcaccaccat ggtaggcccc tatcctacca 1320
tcgaaagttg atagggcaga aatttgaatg atgcgtcgcc ggcacgaagg ccgtgcgatc 1380
cgtcaagtta tcatgaatca tcggatcggc gggcagagcc cgcgtcagcc ttttatctaa 1440
taaatgcgcc cctcccggaa gtcggggttt gttgcacgta ttagctctag aattactacg 1500
gttatccgag tagcacgtac catcaaacaa actataactg atttaatgag ccattcgcag 1560
tttcacagtt cgaattagtt catacttgca catgcatggc ttaatctttg agacaagcat 1620
atgactactg gcaggatcaa ccaggtaccc aa 1652
<210> 19
<211> 10674
<212> DNA
<213> maize
<220>
<221> features not yet classified
<222> (3089)..(3188)
<223> n is a, c, g, or t
<400> 19
ccgacgccgc cggagatttt atctcgccgc cgttccacac acaccgcgac gtggacagcc 60
agcaccgctg ttattcttga accacggtat gagttcgttt gcttgcaatt gcaatagtcc 120
agcttctagt ttgttcgatc tatgtgcgta ttggcctgtg gtagtttctt tcttaggccg 180
tcgtttgggc ggggcgcgcc aggaaggagc agtctgcatc taataatcac ttgaacccaa 240
ttcaattcag tatatacaat tcttcttata tagacggaga catccatcta ctttcatcta 300
acatcatttt acacctgttt tgcttaaatt ctagatatac ttattacact tagcttgtga 360
ttcaacaggt accgtaccgt catgggcatg ttgatgaaca acgacgacag cagcagcagc 420
aggactagca tgcatccacg gccgcaggtc cttgcttccc tgcccttgct ggtctacgaa 480
tacgaggatc atcccaatgc cacaacaagg atgctcatat atagcctacc cgagcggagc 540
attgtctaca cgcacaacag cagtaggccc cagatgatga tgatggaggg taacttatcc 600
ttcagcaccc ctcaaggatg gctggtcatc cttggacaag cgtcagaggc ctcgatctgg 660
catccgctca ccggagagac catcacgctc ccaccaatac acggcgacca ccgtatcccc 720
gatagctgca agtgtctgct cacccgcagc tccgtcgccc acccggactg cgccgtcgtg 780
cttctcgacg tcaacgatcc tctcatgtgg ttctgccggg tgaatggcgg ggccgacagg 840
atgtgggtgc agcacgccta cgacattggc gaccactact tccccgagga gttccgcact 900
ccctccacgc ccaccaagaa tgttgtcgac gacgtcgccg cgctgggagg gaagctgtat 960
tttcgcttca ccgaatcaga ccaagacttc atgggcgtct ttgacttcga tttccatggc 1020
catactcccg ccgtggagtt ttacgagttt gatgtctccg aagagttcaa cctcaagttt 1080
cccgagggcg tgtgctctgc ctccatccac ttggtggagt ccatggatga gctttttgct 1140
gtctgcatct tctatgtcga ttttgatccc accaacatta gcgccgctca tatcttcaag 1200
atggaggaaa tctgcgacga ggaacccgtg gcctggcacc gggtggatga tattggcgac 1260
agggctttcc tcctgacggg caccaacatg tcaacttggt gctctgcaag cacgaataac 1320
ctgaaaggga actccctcta ctttctaggc cacttagtag ctggccacag gaatctctgc 1380
atctatgata ttcaggagca atccatggag attgtccagg ttcacgacca agaagatatg 1440
gagatcgtgc gcacaccgcc atactggatt aatgtacctc cgtgctagta ttagcctatt 1500
acaatgtaat ttgcttaatt aatgtagctt gcttgctagc tagtgctatt tactgctgcc 1560
actactatat agtatattga atcaataaaa atagagtttg ctcgggtttt tgcattatcc 1620
gtgtatgaag ttttagaaga ggatttagca tctcaatttt tttttgggga caatgtacat 1680
catctatagg tatgtgtttg cactgaataa ggggtgattg gttctgtagc acaggtcttc 1740
ttccattgtg gcctaaaaga ctcagcaata ccagccctgt ctctataagc atgtcatatt 1800
caaggagata atatactttt tattactctc ttcctttttt cggtggtaaa aaaacccctc 1860
ccatctcttc tttaataatt agagggggct acaatttctc gtttcgttaa tgtctactgt 1920
agtgcacagt ctctgtgatt tggtacagta ctgttttctg aaatctcatg gtttgttcat 1980
atcagtggtg atgcggtacc tctgtgttat gtgctagcgc ttgtttcatt agctagcctt 2040
agcttaaggc gtccagtaaa tattatattg tctgtgaatt ttatgtgctt caggttggtg 2100
tattattggt gctagattct gaagaacttg ccacaaaatg gagattgtcc aggttcacga 2160
ccagaaagat ttggaaatcg tgcgcacaca gccatcctgg attgtcgata ccgccatgct 2220
aagtcgatta caatgtactt gcttaattaa tgtaggttgc tactagtgat tgcgctaatt 2280
cggccggctg cttctctcat ggtcgtccgc tcacttggga actgacgttt acaagacaga 2340
actacttcta ctagggataa caagcacggg tgaactgttg ttgaggccgg cttgcagtca 2400
tcctgtagcg aagcgtcaat cacttgcagt agtatctcgt ctgggaagct cctctcgacc 2460
aagctagtaa tgctgagtac gttttcaatc atagggtccg ttggtctctt cccggtcaac 2520
atctgtaaaa gtaaaactcc aaaactgtac acatccaagt gaggttgtgc tgtatgctgc 2580
gtctcgtcca gtcgtcgttg tggtacgata gaagctcgca atgccgaagt cccctagacg 2640
agcgttcatg tcatatcatc gaggaggaca ttactcggct tcacatcacc ttcaaatcta 2700
ttccccgcta gggtcagaga tgacaaccga gtaagatcca atggcggacc taggtttttt 2760
tttttttgta tagggtatgc ctcaataaaa cttttacata caattctata taatatactc 2820
catcggttcc aaaatagtat tacttttagc tcttggcttt tatgtcaaca ttcaaatgta 2880
tagcgatgaa tctagacaca taaaatacat acaacaaaca ttttatgaac caattaatta 2940
cctaaaacga attttaattt aggatagaga gagagtacat ataattttat agtaaattca 3000
ataaccaact cggtaaatag aaataaagtc atagtacatc aactagtaac aagacaacaa 3060
ttaacaacgc catctaagtc gctgcatann nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3120
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3180
nnnnnnnnat gtcttggtga agggcacggt ggaacggagg gaaaacgaca cggaggcacg 3240
cgacgacggg cggcaggggc gttcggcctt gcgtctgggc tggaaacgag gggttcgcca 3300
tggcgcgcgg gccgaaaacg gaggcttggg cacgaactcg aaaataagct aagtccaagc 3360
gtgtggaaag acaccgaacc taaagtgcat gtcttgagtg aagggcaagg tggaacggag 3420
ggaaaattgg aggaggcgcg cctcgacggg cggcagggcc gttcggcctt gcgtattggc 3480
tgaaaacgag gggttcgcca tggcgcgcgg gccgaaaaaa acggttcggc cacggccgcg 3540
aaaacgagct aagtcccggc gtgtggaaag acaccgaacc tagagcgcat gtctggagtg 3600
aaggtgaagg tggaacggag ggaaaacggg aggaggcgcg cggcgacggg cggcagggct 3660
gttcggcctt gcgtatgggc tgaaaacgag gtgttcgcca tggcgcgcgg gccgaaaaca 3720
acggttcggc cacggccgcg aaaacgggct aagtccaagc gcgtggaaag acaccgaggc 3780
tgctacgtag gtctcggttg aagggcacgg tggaacggag gaaaaacggg aggagacgca 3840
aggcaacagg cggcagggct gttcggcctt gcgttttggg tgaaaacgag gggttcgcca 3900
tggcgaacgg gccaaaaacg gatttttggc cacggccgcg aaaacgggca cgtggaaggg 3960
cacaggaccc tcccgtggtc accccgcgtg agacgtggaa gttcgggtcc acccgtgagg 4020
gaaaacgggt cacgctagcg aaacccctga ttctgagcaa aaacgctgtg tccagccatc 4080
ttagatgggt ttcgtggggt actgggaaaa tgccctggtt ttctgaaggc agaactcccc 4140
gaagtcctgg gagggggtat gcccctcagg tatagtaggg ggtagggaac tcgtgcagcc 4200
gaaacccggg cgaaacgctg ctgaaaccat agcgtttcca gcgtctcctc caggtctcct 4260
cggccgagcc ccgcaccccc tcgcggccta gccgtggccg gtcggcaccc taccgcgccc 4320
agctccggct ggcgctgttg gctgcctggc cggccgtggt tcggccgtga cgcagccacg 4380
accggctatg gctggctacc gccggccaga cgccctggac gagtggctgc accgtgggat 4440
ggcccgttgc tcgatgcgtt ttccgtttct cccgcgctcg gtggaccttc ggtcgccgtc 4500
ctcgcaagca gacccgccgc gcccagcgcg gagggatgct ttggatggcc cgaccgtagc 4560
ggctacgctg gcgcatgagt tgtcttggac ccgtgactgc ttggaggacc cccgctgccg 4620
tgcggccgac tcccggcgcc cgtgtcccat cgctcgtgcg ggcatcctgt gcctgctgcg 4680
ttgagaagtg cttgcgtgct gctacccgtc ccacgggaag ccgtgctcga tacacgttgc 4740
cttcgtcgag ctcacccccc ggggtgcggc tcgtcggctc gagagcgccc gcggcgtttg 4800
cctcgtgccg ccgtcggcct atggccggcg gcaccgagga cacctcgctg gcgcttttgg 4860
tctcggatgt ggctcacgct gaaggccgga gacgcgttgg cgtcacgcgc ccaagaatcg 4920
gtccgcccga atgaacgacg gccagcccgg cacgacgcct ccgcgcggag gccggcgctg 4980
gcccgtctgc gaggacgtgc tacctggttg atcctgccag tagtcatatg cttgtctcaa 5040
agattaagcc atgcatgtgc aagtatgaac taattcgaac tgtgaaactg cgaatggctc 5100
attaaatcag ttatagtttg tttgatggta cgtgctactc ggataaccgt agtaattcta 5160
gagctaatac gtgcaacaaa ccccgacttc cgggaggggc gcatttatta gataaaaggc 5220
tgacgcgggc tctgcccgcc gatccgatga ttcatgataa cttgacggat cgcacggcct 5280
tcgtgccggc gacgcatcat tcaaatttct gccctatcaa ctttcgatgg taggataggg 5340
gcctaccatg gtggtgacgg gtgacggaga attagggttc gattccggag agggagcctg 5400
agaaacggct accacatcca aggaaggcag caggcgcgca aattacccaa tcctgacacg 5460
gggaggtagt gacaataaat aacaataccg ggcgcgttag tgtctggtaa ttggaatgag 5520
tacaatctaa atcccttaac gaggatccat tggagggcaa gtctggtgcc agcagccgcg 5580
gtaattccag ctccaatagc gtatatttaa gttgttgcag ttaaaaagct cgtagttgga 5640
ccttgggccg ggccggccgg tccgcctcac ggcgagaacc gaccggctcg acccttctgc 5700
cggcgatgcg ctcctggcct taactggccg ggtcgtgcct ccggcgccgt tactttgaag 5760
aaattagagt gctcaaagca agccatcgct ctggatacat tagcatggga taacatcata 5820
ggattccggt cctattgtgt tggccttcgg gatcggagta atgattaata gggacagtcg 5880
ggggcattcg tatttcatag tcagaggtga aattcttgga tttatgaaag acgaacaact 5940
gcgaaagcat ttgccaagga tgttttcatt aatcaagaac gaaagttggg ggctcgaaga 6000
cgatcagata ccgtcctagt ctcaaccata aacgatgccg accagggatc agcgggtgtt 6060
actaatagga ccccgctggc accttatgag aaatcaaagt ctttgggttc cggggggagt 6120
atggtcgcaa ggctgaaact taaaggaatt gacggaaggg caccaccagg cgtggagcct 6180
gcggcttaat ttgactcaac acggggaaac ttaccaggtc cagacatagc aaggattgac 6240
agactgagag ctctttcttg attctatggg tggtggtgca tggccgttct tagttggtgg 6300
agcgatttgt ctggttaatt ccgttaacga acgagacctc agcctgctaa ctagctatgc 6360
ggagccatcc ctccatagtt agcttcttag agggactatg gccgtttagg ccacggaagt 6420
ttgaggcaat aacaggtctg tgatgccctt agatgttctg ggccgcacgc gcgctacact 6480
gatgtatcca acgagtatat agccttggcc gacaggcccg ggtaatcttg ggaaatttca 6540
tcgtgatggg gatagatcat tgcaattgtt ggtcttcaac gaggaatgcc tagtaagcgc 6600
gagtcatcag ctccgttgac tacgtccctg ccctttgtac acaccgcccg tcgctcctac 6660
cgattgaatg gtccggtgaa gtgttcggat cacggcgacg ggggcggttc gccgcccccg 6720
acgtcgcgag aagtccattg aaccttatca tttagaggaa ggagaagtcg taacaaggtt 6780
tccgtaggtg aacctgcgga aggatcattg ccgtgaccct taaacaaaac agaccgcgaa 6840
cgagtcaccc gtgccgccgg gctccggccc ggcacgctgc cccccccgaa cctcccgcgg 6900
ggaagggggg tgccgcgaaa aagaacccac ggcgccccgg gcgccaagga acaccagtac 6960
tacctcctgc cccgcggagc ggtcggcccg ccttccgctc ccagggcagc ggttacacct 7020
taatcgacac gactctcggc aacggatatc tcggctctcg catcgatgaa gaacgtagca 7080
aaatgcgata cctggtgtga attgcagaat cccgcgaacc atcgagtttt tgaacgcaag 7140
ttgcgcccga agccttctgg cggagggcac gtctgcctgg gcgtcacgcc aaaagacact 7200
cccaacaccc ccccgcgggg cgagggacgt ggcgtctggc cccccgcgcc gcagggcgag 7260
gtgggccgaa gcaggggctg ccggcgaacc gcgccgggcg cagcacgtgg tgggcgacat 7320
caagttgttc tcggtgcagc gtcacggcgc gcggccggac attcggccct aaggacccat 7380
cgagcgaccg agcttgccct cggaccgcga ccccaggtca gtcgggacta cccgctgagt 7440
ttaagcatat aaataagcgg aggagaagaa acttacgagg attcccctag taacggcgag 7500
cgaaccggga gcagcccagc ttgagaatcg ggcggcctcg ccgcccgaat tgtagtctgg 7560
agaggcgtcc tcagcgacgg accgggccca agttctctgg aaagggacgc ctgggagggt 7620
gagagccccg tccggcccgg accctgtcgc accacgaggc gccgtcaacg agtcgggttg 7680
tttgggaatg cagcccaaat cgggcggtaa actccgtcca aggctaaata caggcgagag 7740
accgatagcg aacaagtacc gcgagggaaa gatgaaaagg actttgaaaa gagagtcaaa 7800
gagtgcttga aattgccggg agggaagcgg atgggggctg gcgacgcgca ccggccgtat 7860
gcggaacggc tcctgctggt ccgccgatcg gctcggggcg tggaccgttg tcgcccgcgc 7920
cggcggccaa agcccggggg ccctaggcgc ccccggcagc cgtcgtcggc gcggacggta 7980
tccgcgcgcc tctggcgcgc ccctcggggc gctgcgccgc aacggcctgc gagctcccca 8040
tccgacccgt cttgaaacac ggaccaagga gtctgacatg cgtgcgagtc gacgggttca 8100
gaaacctgag atgcgcaagg aagctgacga gcgggaggcc ctcacgggcc gcaccgctgg 8160
ccgaccctga tcttctgtga agggttcgag ttggagcacg cctgtcggga cccgaaagat 8220
ggtgaactat gcctgagcgg ggcgaagcca gaggaaactc tggtggaggc tcgaagcgat 8280
actgacgtgc aaatcgttcg tctgacttgg gtataggggc gaaagactaa tcgaaccatc 8340
tagtagctgg ttccctccga agtttccctc aggatagctg gagcccacac gagttctatc 8400
gggtaaagcc aatgattaga ggcatcgggg gcgcaacgcc ctcgacctat tctcaaactt 8460
taaataggta ggacggcgcg gctgcttcgg tgagccgtgc cacggaatcg ggagctccaa 8520
gtgggccatt tttggtaagc agaactggcg atgcgggatg aaccggaagc cgggttacgg 8580
tgccaaactg cgcgctaacc tagaacccac aaagggtgtt ggtcgattaa gacagcagga 8640
cggtggtcat ggaagtcgaa atccgctaag gagtgtgtaa caactcacct gccgaatcaa 8700
ctagccccga aaatggatgg cgctgaagcg cgcgacccac acccggccat ctgggcgagc 8760
gacatgcccc gatgagtagg agggcgcggc ggccgccgca aaacccgggg cgcgagcccg 8820
ggcggagcgg ccgtcggtgc agatcttggt ggtagtagca aatattcaaa tgagaacttt 8880
gaaggccgaa gaggagaaag gttccatgtg aacggcactt gcacatgggt aagccgatcc 8940
taagggacgg gggaaacccg gcagatagcg cgatcacgcg cgtcacccga aagggaatcg 9000
ggttaagatt tcccgagccg ggacgtggcg gcagacggcg acgttaggaa gtccggagac 9060
gccggcgggg gcctcgggaa gagttatctt ttctgcttaa cggcccgcca accctggaat 9120
cggttcagcc ggaggtaggg tccagcggcc ggaagagcac cgcacatcgc gcggtgtccg 9180
gtgcgccccc ggcggccctt gaaaatccgg aggaccgaat accgtccacg cccggtcgta 9240
ctcataaccg catcaggtct ccaaggtgaa cagcctctgg ccaatggaac aatgtaggca 9300
agggaagtcg gcaaaacgga tccgtaactt cgggaaaagg attggctctg agggttgggc 9360
tcgggggtcc cggccccgaa cccgtcggct gctggcggaa tgctcgagct gctcgcgcgg 9420
cgagagcggg ccgccgcgtg ccggccgggg gacggaccgg gaacggcccc ctcgggggcc 9480
ttccccgggc gtcgaacaac cgactcagaa ctggtacgga caaggggaat ccgactgttt 9540
aattaaaaca aagcattgcg atggtcctcg aggatgctga cgcaatgtga tttctgccca 9600
gtgctctgaa tgtcaaagtg aagaaattca accaagcgcg ggtaaacggc gggagtaact 9660
atgactctct taaggtagcc aaatgcctcg tcatctaatt agtgacgcgc atgaatggat 9720
taacgagatt cccactgtcc ctgtctacta tccagcgaaa ccacagccaa gggaacgggc 9780
ttggcggaat cagcggggaa agaagaccct gttgagcttg actctagtcc gactttgtga 9840
aatgacttga gaggtgtagg ataagtggga gcctccgggc gcaagtgaaa taccactact 9900
tttaacgtta ttttacttat tccgtgggtc ggaagcgggg caccgcccct ccttttggct 9960
ccaaggcccg gcctcgccgg gccgatccgg gcggaagaca ttgtcaggtg gggagtttgg 10020
ctggggcggc acatctgtta aaagataacg caggtgtcct aagatgagct caacgagaac 10080
agaaatctcg tgtggaacaa aagggtaaaa gctcgtttga ttctgatttc cagtacgaat 10140
acgaaccgtg aaagcgtggc ctatcgatcc tttagacctt cggagtttga agctagaggt 10200
gtcagaaaag ttaccacagg gataactggc ttgtggcagc caagcgttca tagcgacgtt 10260
gctttttgat ccttcgatgt cggctcttcc tatcattgtg aagcagaatt caccaagtgt 10320
tggattgttc acccaccaat agggaacgtg agctgggttt agaccgtcgt gagacaggtt 10380
agttttaccc tactgatgac cgcgccgcga tagtaattca acctagtacg agaggaaccg 10440
ttgattcaca caattggtca tcgcgcttgg ttgaaaagcc agtggcgcga agctaccgtg 10500
tgccggatta tgactgaacg cctctaagtc agaatccaag ctagcaaccg gcgcctctgc 10560
tcgccgcccg ccccgaccca cgttagggcg ttcgcgcccc aagggcccgt gccattggct 10620
cagcccgccc ggccgacgcg ccgcggcggg ccgcctcgaa gctcccttcc caac 10674
<210> 20
<211> 674
<212> DNA
<213> maize
<400> 20
tacctggttg atcctgccag tagtcatatg cttgtctcaa agattaagcc atgcatgtgc 60
aagtatgaac taattcgaac tgtgaaactg cgaatggctc attaaatcag ttatagtttg 120
tttgatggta cgtgctactc ggataaccgt agtaattcta gagctaatac gtgcaacaaa 180
ccccgacttc cgggaggggc gcatttatta gataaaaggc tgacgcgggc tctgcccgcc 240
gatccgatga ttcatgataa cttgacggat cgcacggcct tcgtgccggc gacgcatcat 300
tcaaatttct gccctatcaa ctttcgatgg taggataggg gcctaccatg gtggtgacgg 360
gtgacggaga attagggttc gattccggag agggagcctg agaaacggct accacatcca 420
aggaaggcag caggcgcgca aattacccaa tcctgacacg gggaggtagt gacaataaat 480
aacaataccg ggcgcgttag tgtctggtaa ttggaatgag tacaatctaa atcccttaac 540
gaggatccat tggagggcaa gtctggtgcc agcagccgcg gtaattccag ctccaatagc 600
gtatatttaa gttgttgcag ttaaaaagct cgtagttgga ccttgggccg ggccggccgg 660
tccgcctcac ggcg 674
<210> 21
<211> 721
<212> DNA
<213> maize
<400> 21
tacctggttg atcctgccag tagtcatatg cttgtctcaa agattaagcc atgcatgtgc 60
aagtatgaac taattcgaac tgtgaaactg cgaatggctc attaaatcag ttatagtttg 120
tttgatggta cgtgctactc ggataaccgt agtaattcta gagctaatac gtgcaacaaa 180
ccccgacttc cgggaggggc gcatttatta gataaaaggc tgacgcgggc tctgcccgcc 240
gatccgatga ttcatgataa cttgacggat cgcacggcct tcgtgccggc gacgcatcat 300
tcaaatttct gccctatcaa ctttcgatgg taggataggg gcctaccatg gtggtgacgg 360
gtgacggaga attagggttc gattccggag agggagcctg agaaacggct accacatcca 420
aggaaggcag caggcgcgca aattacccaa tcctgacacg gggaggtagt gacaataaat 480
aacaataccg ggcgcgttag tgtctggtaa ttggaatgag tacaatctaa atcccttaac 540
gaggatccat tggagggcaa gtctggtgcc agcagccgcg gtaattccag ctccaatagc 600
gtatatttaa gttgttgcag ttaaaaagct cgtagctcga cccttctgcc ggcgatgcgc 660
tcctggcctt aactggccgg gtcgtgcctc cggcgccgtt actttgaaga aattagagtg 720
c 721
<210> 22
<211> 10611
<212> DNA
<213> maize
<400> 22
accacataaa aacattcccc ctagagtagc tgttaatacg aataacagaa actctgttat 60
agccatttct gtacattcaa tgtactctac ggatagagga atacataaag ttgaacataa 120
taaaataaga aattgaaaga tttcgttgaa attgttcgtt tggaaatttc ccgaaaagct 180
aattataggt tcttctctcc atcggaacaa tagggccgtt atgcttatta ctaaacttgt 240
tgaagagatg aaatagaacc aaggtctatc tttttgatca gaggttaaat cgatcatcag 300
aagaagaatt aggccaaaaa ttaggataca ttctgggaaa atgaaacttc catggaagag 360
aagcaaatga aacgctttca taaaaattct cgtagaatcg agaatgaagt tttcattctg 420
tacatgccag atcatgaatt agtaactgca gccaatctcc gaaaagtccc gattgtttcg 480
atttttggaa tgggatattt acggaatccc catgaatagg atcaaacctt attccatgct 540
atttccataa gattcctctt tcttattctt aagcaagccc ccgagagggc ttagttgatc 600
atgatttctg ttttctcttt tttttccttt ttatttgttt cgaaaaagat atcgtccgat 660
tctccttcta ttgattcttt tccgatcgag atgtatggat ccatgtgtct acatacctag 720
attctgttca tggattaacg aaaatgtgca agagctctat ttgcctctgc cattctatga 780
gtcgcttcct ttttgcgtat ggcaccccca ctccctttgg cagcatctac taattcggaa 840
cttaatttga aagccatatt tcgacccgga cgcttttggg atgcttctaa taaccaacga 900
atggcaagtg ctcttccttg tttagatcct atttcaatcg gaactttccg cgtcgatcct 960
tttttattac gtcttgtttt tactcctata ttgggagtta ctctacgtat tgcttgacgt 1020
aaaaccaata gtggatttgt ttctgtcttt tgttgaatct ttttcacggc tcgatagaga 1080
atttgataag ccaatgattt ttttccgtct ttcataatac ggttaaccac catgttaact 1140
aatcgattac gaaaaattgg atcggatttt gcggttcttt tttctgcagt acctcgacgt 1200
gacatgagcg tgaaagaggt tcaagaatcc gttttctttt tataagggct aaaaacgaat 1260
cacttatttt tttggctttt tggccccata ttgtagggtg gatctcgaaa gataggaaag 1320
atctccctcc aagccgtaca tacgactttc atcgaatacg gctttccaca gaattctata 1380
gggatctatg agatcgagta tggaattctg tttactcact ttaaattgag tatccgtttc 1440
cctccttttc ccgctaggac cggaaatcct gtattttcca tatccatacg atcgagtcct 1500
taggtttccg aaatagtgta atggaaaaag aagtgcttcg aatcattgct atttgactcg 1560
gacctgttct gaaaaagtcg aggtatttcg aattgtttgt tgacacggac aaagtaaggg 1620
aaaacctctg aaagaatttc catattgacc ttggacatat aagagttccg aatcgaatct 1680
ctttagaaag aagatctttt gtctcatggt agcctgctcc agtcccctta cgaaactttc 1740
gttattgggt tagccataca cttcacatgt ttctagcgat tcacatggca tcatcaaatg 1800
atacaagtct tggataagaa tctacaacgc actagaacgc ccttgttgac gattctttac 1860
tgcgacagca tctagggttc ctcgaataat gcgatatctc acaccgggta aatccttaac 1920
ccttcctcct cttactaata ctacagaatg ttcttgtaaa ttatggccaa taccaggtat 1980
ataagcagtg atttcaaatc cagaggttaa tcgtactctg gcaactttac gtaaggcaga 2040
gttgggtttt ttggggttga tagtggaaaa gtcgacagat aagtcatcct tactgtccct 2100
ctacagaacc gtacatgaga ttttcacctc atacggctcc tcgttcaatt ctttcgaagg 2160
gatccttttc ctcgttcgag agtctccgcc cttcttccac tccgtcccga agactaacta 2220
agaccaattg agtcacgttt tcatgttcta attgaacact ttccatttat gattaaagga 2280
gaagattgtt cttttaccaa acatatgcgg atcaaatcac gtcttataat aagaagaaat 2340
ctttctcggt atcaatcccc ttgcccctca ttctttgaga atcagaagga tccttttcga 2400
gtttccattt cttcattttg aatctgggct cttctatctt cgacttattt ttttggcttt 2460
attctttatt tatttcattt cgatttttcc ctcttcctct atccctatcc tctaggtaca 2520
gcgtttgcat caatagagaa ctttttcctc tgtatgaatc gatattattc caatttcttc 2580
ccgaaacttc ccaagaaaaa tcccgaattg gatccaaaat tgacgggtta atgtgagctt 2640
atccatgcgg ttaggcactc ttcaaatagg aatccatttt ctaactggct ttcgtgcttt 2700
ggtgagtcgt ccgagatcct ttcgatgacc tatgttgtgt tgaagggata tctatatgat 2760
ccgatcgatt gcataagacc cgcggtagca atagaacggg gaaagtatac agaaaagaca 2820
gttcttttcg atttcgatta tctatatatt agttcgtttc tatttctaga tatctatttc 2880
tatatatcta tatattagta ttaatatcta tatattagta ttagttatct atatattagt 2940
attagttagt agtactattc tattagttag cgatcccggc tctgtgagtt ctttcttccg 3000
tgatgaactg tcggcaccag tcctacattt tttctctgtg gaccgaggag aaagggggct 3060
cagcaggaag aggattgtac catgagagaa gcacagaggt caacccgctt caaatatgga 3120
acatggattc tggcaatgca acggagttgg gtcctcatat cgatccgaat gaatcagtct 3180
ttctacagag gtcaatcttt gcctattagg caagaggata gcaagttcga aattctgtct 3240
cggtaggaca tggatttcta ttactatgaa attcataaat tagttaatgg gggggctacc 3300
attatccttt ttcttgtatg tgttcctaag agaaggaatt tgtccatttc atgtttcgag 3360
gtctcaaaaa aagggcgtgg aaacagatag aaactcttga atggaaattg aaaagaaatg 3420
tagccccagt tccttcggaa atggtaagat ctttggcgca agaagaaggg gcgatccata 3480
tcatcttgac ttggttctgc ttcccctctt tttttaagaa taccgagtcg ggttcttctc 3540
ctaccagtat cgaatagaac atgctgaaca agatcttctt catggaaacc cactcgattt 3600
agatcgggaa aatcgtacag attttatgaa accatgtgct atggctcgaa tccatagtca 3660
atcctatttt cgataggacc ggttgacaat tgaatccaat ttttcccatt atttgactgt 3720
ccataatagt gcggaaagaa agcccggagg aagagtggcc ttgcgtttct cgcccctttg 3780
ccttaggatt cgttaattct ctttctcgat gggacgggga agggatataa ctcagcggta 3840
gagtgtcacc ttgacgtggt ggaagtcatc agttcgagcc tgattatccc taaacctaat 3900
gtgagttttt tctattttga cttactcccc caccacgatc gaacgggaat ggataagagg 3960
cttgtgggat tgacgtgata gggtagggtt ggctatactg ctggtggcga actccaggct 4020
aataatctga agcgcatgga tacaagttat ccttggaagg aaagacaatt ccgaatccgc 4080
tttgtctacg aataaggaag ctataagtaa tgcaactatg aatctcatgg agagttcgat 4140
cctggctcag gatgaacgct ggcggcatgc ttaacacatg caagtcgaac gggaagtggt 4200
gtttccagtg gcgaacgggt gagtaacgcg taagaacctg cccttgggag gggaacaaca 4260
actggaaacg gttgctaata ccccgtaggc tgaggagcaa aaggagaaat ccgcccaagg 4320
aggggctcgc gtctgattag ctagttggtg aggcaatagc ttaccaaggc gatgatcagt 4380
agctggtccg agaggatgat cagccacact gggactgaga cacggcccag actcctacgg 4440
gaggcagcag tggggaattt tccgcaatgg gcgaaagcct gacggagcaa tgccgcgtgg 4500
aggtggaagg cctacgggtc gtcaacttct tttctcggag aagaaacaat gacggtatct 4560
gaggaataag catcggctaa ctctgtgcca gcagccgcgg taagacagag gatgcaagcg 4620
ttatccggaa tgattgggcg taaagcgtct gtaggtggct tttcaagtcc gccgtcaaat 4680
cccagggctc aaccctggac aggcggtgga aactaccaag ctggagtacg gtaggggcag 4740
agggaatttc cggtggagcg gtgaaatgca ttgagatcgg aaagaacacc aacggcgaaa 4800
gcactctgct gggccgacac tgacactgag agacgaaagc taggggagca aatgggatta 4860
gagaccccag tagtcctagc cgtaaacgat ggatactagg tgctgtgcga ctcgacccgt 4920
gcagtgctgt agctaacgcg ttaagtatcc cgcctgggga gtacgttcgc aagaatgaaa 4980
ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgatgcaa 5040
agcgaagaac cttaccaggg cttgacatgc cgcgaatcct cttgaaagag aggggtgccc 5100
tcgggaacgc ggacacaggt ggtgcatggc tgtcgtcagc tcgtgccgta aggtgttggg 5160
ttaagtctcg caacgagcgc aaccctcgtg tttagttgcc actatgagtt tggaaccctg 5220
aacagaccgc cggtgttaag ccggaggaag gagaggatga ggccaagtca tcatgcccct 5280
tatgccctgg gcgacacacg tgctacaatg ggcgggacaa agggtcgcga tctcgcgagg 5340
gtgagctaac tccaaaaacc cgtcctcagt tcggattgca ggctgcaact cgcctgcatg 5400
aagcaggaat cgctagtaat cgccggtcag ccatacggcg gtgaatccgt tcccgggcct 5460
tgtacacacc gcccgtcaca ctataggagc tggccaggtt tgaagtcatt acccttaacc 5520
gtaaggaggg ggatgcctaa ggctaggctt gcgactggag tgaagtcgta acaaggtagc 5580
cgtactggaa ggtgcggctg gatcacctcc ttttcaggga gagctaatgc ttatgcttat 5640
tgggtatttt ggtttgacac tgcttcacgc ccaaaaagaa ggcagctacg tctgagctaa 5700
acttggatat ggaagtcttc tttcgtttag ggtgaagtaa gaccaagctc atgagcttat 5760
tatcctaggt cggaacaaat tagttgatag tgataggatc ccctttttga cgtccccatg 5820
tccccccgtg tggcggcatg gggatgtcaa aaggaaaggg atggagtttt tctcgctttt 5880
ggcgtagcag gcctcccttt gggaggcccg cgcgacgggc tattagctca gtggtagagc 5940
gcgcccctga taattgcgtc gttgtgcctg ggctgtgagg gctctcagcc acatggatag 6000
ttcaatgtgc tcatcagcgc ctgacccgaa gatgtggatc atccaaggca cattagcatg 6060
gcgtactcct cctgtttgaa tcggagtttg aaaccaaaca aacttctcct caggaggata 6120
gatggggcga ttcaggtgag atcccatgta gatcgaactt tctattcact cgtgggatcc 6180
gggcggtccg ggggggggcc accgcggctc ctctcttctc gagaatccat acatccctta 6240
tcagtgtatg gagagctatc tctcgagcac aggttgaggt tcgtcctcaa tgggaaaatg 6300
gagcacctaa caacgcatct tcacagacca agaactacga gatcacccct ttcattctgg 6360
ggtgacggag ggatcgtacc attcgagcct ttttttcatg cttttcccgg cggtctggag 6420
aaagcagtaa tcaataggac ttccctaatc ctcccttcct gaaaggaaga acgtgaaatt 6480
ctttttcctt tctgcaggga ccaggagatt ggatctagcc ataagaggaa tgcttggtat 6540
aaataagcca cttcttggtc ttcgaccccc taagtcacta cgagcgcccc cgatcagtgc 6600
aatgggatgt ggctatttat ctatctcttg actcgaaatg ggagcagagc aggtttgaaa 6660
aaggatctta gagtgtctag ggttgggcca ggagggtctc ttaacccctt cttttttctg 6720
cccatcggag ttatttccca aggacttgcc gtggtaaggg ggagaagggg gaagaagcac 6780
acttgaagag cgcagtacaa cggggagttg tatgctgcgt tcgggaagga tgaatcgctc 6840
ccgaaaagga gtctattgat tctctcccaa ttggttggat cgtaggggcg atgatttact 6900
tcacgggcga ggtctctggt tcaagtccag gatggcccag ctgcgccagg gaaaagaata 6960
gaagaagcat ctgactcttt catgcatact ccacttggct cgggggggat atagctcagt 7020
tggtagagct ccgctcttgc aattgggtcg ttgcgattac gggttggctg tctaattgtc 7080
caggcggtaa tgatagtatc ttgtacctga accggtggct cactttttct aagtaatggg 7140
gaagaggact gaaacatgcc actgaaagac tctactgaga caaaaagatg ggctgtcaaa 7200
aaggtagagg aggtaggatg ggcagttggt cagatctagt atggatcgta catggacgat 7260
agttggagtc ggcggctctc ctaggcttcc ctcatctggg atccctgggg aagaggatca 7320
agttggccct tgcgaatagc ttgatgcact atctcccttc aaccctttga gcgaaatgtg 7380
gcaaaaggaa ggaaaatcca tggaccgacc ccattgtctc caccccgtag gaactacgag 7440
atcaccccaa ggacgccttc ggcgtccagg ggtcacggac cgaccataga tcctgttcaa 7500
taagtggaac acattagccg tccgctctcc ggttgggcag taagggtcgg agaagggcaa 7560
tcactcgttc ttaaaaccag cattcttaag ttaagatcaa agagtcgggc ggaaaaaggg 7620
gagagctccc cgttcctggt tctcctgtag ctggattccc cggaaccaca agaatcctta 7680
gaatgggatt ccaactcagc accttttgtt ttgggatttt gagaagagtt gctctttgga 7740
gagcacagta cgatgaaagt tgtaagctgt gttcgggggg gagttattgc ctatcgttgt 7800
cctctatggt agaacccgtc ggggaggcct gagaggcggt ggtttaccct gtggcggatg 7860
tcagcggttc gagtccgctt atctccagcc cgtgaactta gcggatacta tgatagcacc 7920
gaaggttgcc aattcgtcag ttcgatctat gatttcgcat tcatggacgt tgataagatc 7980
cttccattta gtagcacctt aggatggcat agccttaacg ttaatggcga ggttcaaaag 8040
aggaaaggct tgcggtggat acctaggcac ccagagacga ggaagggcgt agcaagcgac 8100
gaaatgcttc ggggagttga aaataagcat agatccggag attcccaaat aggtcaacct 8160
tttgaactgc ctgctgaatc catgagcagg caagagacaa cctggcgaac tgaaacatct 8220
tagtagccag aggaaaagaa agcaaaagcg attcccgtag tagcggcgag cgaaatggga 8280
gcagcctaaa ccgtgaaaac ggggttgtgg gagagcaata caagcgttgt gctgctaggc 8340
gaagcggttg agtgccgcac cctagatggc taaagtccag tagccgaaag catcactagc 8400
ttacgctctg acccgagtag catggggcac gtggaatccc gtgtgaatca gcaaggacca 8460
ccttgcaagg ctaaatactc ctgggtgacc gatagcgaag tagtaccgtg agggaaaggt 8520
gaaaagaacc cccagtgggt agtgaaatag aacgtgaaac cgtgctgagc tcccaagcag 8580
tgggagggga aagtgatctc tgaccgcgtg cctgttgaag aatgagccgg cgactcatag 8640
gcagtggctt ggttaaggga atggaaccca ccggagccgt agcgaaagcg agtcttcata 8700
gggcgattgt cactgcttat ggacccgaac ctgggtgatc tatccatgac caggatgaag 8760
cttggatgaa actaagcaga ggtccgaacc gactgatgtt gaagaatcag cggatgagtt 8820
gtggttaggg gtgaaatgcc actcgaaccc agagctagct ggttctcccc gaaatgcgtt 8880
gaggcgcagc agttgactgg acatctaggg gtaaagcact gtttcggtgc gggctgcgcg 8940
agcggtacca aatcgaggca aactctgaat actagatatg acccaaaaat aacaggggtc 9000
aaggtcggcc agtgagacga tgggggataa gcttcatcgt cgagagggaa acagcccgga 9060
tcaccagcta aggcccctaa atgaccgctc agtgataaag gaggtggggg tgcaaagaca 9120
gccaggaggt ttgcctagaa gcagccaccc tttaaagagt gcgtaatagc tcactgatcg 9180
agcgcccttg cgctgaagat gaacggggct aagcgatctg ccgaagctgt gggatgtcaa 9240
aatgcatcgg taggggagcg ttccgcctta gagggaagca aacgcgaaag cgggggtcga 9300
cgaagcggaa gcgagaatgt cggcttgagt aacgaaaaca ttggtgagaa tccaatgccc 9360
cgaaaaccca aggtttcctc cgcaaggttc gtccacggag ggtgagtcag ggcctaagat 9420
caggccgaaa ggcgtagtcg atggacaaca ggtcaatatt cctgtactac cccttgttgg 9480
tacggaggga cggaggaggc taggttagcc gaaagatggt tataggttta aggacacaag 9540
gtgaccctgc tttttcaggg taagaagggg tagagaaaat gcctcgagcc gaggtccgag 9600
taccaagcgc tgcagcgctg aagtatgagc cccgtggact agccattgct tctccacgag 9660
gctcatacca ggcgctacgg cgctgaagta tgtaacccat gccatactcc caggaaaagc 9720
tcgaacgacc ttcaacaaaa gggtacctgt acccgaaacc gacacaggtg ggtaggtaga 9780
gaatacctag gggcgcgaga caactctctc taaggaactc ggcaaaatag ccccgtaact 9840
tcgggagaag gggtgccccc tcgcaaaagg gggtcgcagt gaccaggccc gggcgactgt 9900
ttaccaaaaa cacaggtctc cgcaaagtcg taagaccatg tatgggggct gacgcctgcc 9960
cagtgccgga aggtcaagga agttggtgaa ctgatgacag ggaagccggc gaccgaagcc 10020
ccggtgaacg gcggccgtaa ctataacggt cctaaggtag cgaaattcct tgtcgggtaa 10080
gttccgaccc gcacgaaagg cgtaacgatc tgggcactgt ctcggagaga ggctcggtga 10140
aatagacatg tctgtgaaga tgcggactac ctgcacctgg acagaaagac cctatgaagc 10200
tttactgttc cctgggattg gctttgggct tttcctgcgc agcttaggtg gaaggcgaag 10260
aaggccccct tccggggggg cccgagccat cagtgagata ccactctgga agagctcgga 10320
ttctaacctt gtgtcagacc cgcgggccaa gggacagtct caggtagaca gtttctatgg 10380
ggcgtaggcc tcccaaaagg taacggaggc gtgcaaaggt ttcctcgggc cagacggaca 10440
ttggtcctcg agtgcaaagg cagaagggag cttgactgca agactcaccc gtcgagcaga 10500
gacgaaagtc ggccttagtg atccgacggt gccgagtgga agggccgtcg ctcaacggat 10560
aaaagttact ctagggataa caggctgatc ttccccaaga gtccacatcg a 10611
<210> 23
<211> 611
<212> DNA
<213> maize
<400> 23
cccgcacaag cggtggagca tgtggtttaa ttcgatgcaa agcgaagaac cttaccaggg 60
cttgacatgc cgcgaatcct cttgaaagag aggggtgccc tcgggaacgc ggacacaggt 120
ggtgcatggc tgtcgtcagc tcgtgccgta aggtgttggg ttaagtctcg caacgagcgc 180
aaccctcgtg tttagttgcc actatgagtt tggaaccctg aacagaccgc cggtgttaag 240
ccggaggaag gagaggatga ggccaagtca tcatgcccct tatgccctgg gcgacacacg 300
tgctacaatg ggcgggacaa agggtcgcga tctcgcgagg gtgagctaac tccaaaaacc 360
cgtcctcagt tcggattgca ggctgcaact cgcctgcatg aagcaggaat cgctagtaat 420
cgccggtcag ccatacggcg gtgaatccgt tcccgggcct tgtacacacc gcccgtcaca 480
ctataggagc tggccaggtt tgaagtcatt acccttaacc gtaaggaggg ggatgcctaa 540
ggctaggctt gcgactggag tgaagtcgta acaaggtagc cgtactggaa ggtgcggctg 600
gatcacctcc t 611
<210> 24
<211> 22902
<212> DNA
<213> maize
<400> 24
attcattatt ggcccaccat tgattacaag atttagcttt tatgaatcgc tattggtttg 60
atacgaataa tggcagtcgt ttcagtttgt taaggataca gatgtatcca caattcattt 120
agagttactt aatagcctat ttcttatact atatctctat cccgtgaaat tctcaagccc 180
aaagatggat gcatatgctg tgtttcattt tgctaaatga tatcaattaa atggtatatc 240
aattctataa attggatata acaataaata aatcagaaaa attcttttat tttagataga 300
agaaatgttt cttctatcta aaataaatga atgtaccctt ctatccaaat ccaatttgca 360
tcgataaaat aaatccaaat tccagattct agcagtagat gaataattgc aaatttttgt 420
gtgtacgaga ttagaataac ttaaaaataa ctgacataat tttttatttt tcctgaccaa 480
aaaaatacat gaaaaagaaa ggaggtagaa aaatttgttg atttatggtt aaagaagaaa 540
aacaagaaaa caggggttct gttgaatttc aagtattcag tttcactaat aagatacgga 600
gacttgcttc acatttagaa ttacacaaaa aatatttttc atcggaaaga ggtctacgaa 660
gacttttggg aaaacgtcaa agtttgctgg cttatttggt aaagaaaaat agagtacgtt 720
ataagaaatt aataagtcag ttggatattc gggagaagta atttaatcgt tctcattttt 780
ttcttatttt attagtagtc ttatagtagt attagatttt gtattttgat gagcctcgtt 840
ttgaggaatt catggaataa tccattttca tggaataaag aataagaaca aggatatgag 900
tctatcgctt aaaagaaaag atctcatgat agtcaatatg ggcactcaac acccatcaat 960
gcatggtgtt cttcgactga ttgttactct cgatggtgaa gatgttattg attgtgaacc 1020
catattaggg tatttacaca gaggaatgga aaaaatcgcg gaaaacagaa gtattataca 1080
atacttgcct tatgtaacac gatgggatta tttagctact atgtttacag aagcaataac 1140
ggtaaatgca ccagaattct tagagaatat tcaattaccc aaaagagcca actatattag 1200
agtaattatg ttagaattga gccgtatagc ttctcatttg ttatggcttg gaccttttat 1260
ggaggatctc ggggcacaga ctcccttttt ctacattttt agagagagag agaattgata 1320
tatgatctat ttgaagctgc tacaggtatg cgaatgatgc ataattactt tcgcatcgga 1380
ggagtcgctg ccgatctccc ttatggatgg atggataaat gtttagattt ctgtgattat 1440
tttttacaag gagttgttga atatcaagaa cttattacac agaatcccat ttttttagaa 1500
cgagttgaag gagtcggttt tattagcgga gaagaagctg taaattgggg cttatcggga 1560
ccaatgttac gagcttctgg aatacaatgg gatcttcgta aaattgatcc ttatgagtct 1620
tacaatcaat tcgattggaa agtccaatgg caaaaagaag gagattcgtt agctcgctat 1680
ttagtacgag tcggtgaaat gagggaatcc ataaaaatta ttcaacaggc tgtagagaaa 1740
attcctagag gaccttatga gaatttagaa gcccgacgct ttaagaaagc aaagaatccc 1800
gaatggaatg attttgaata tcgatttctt ggtaaaaaac cttcgcccaa ttctgaatta 1860
tcaaagcaag agctttatgt aagagtagaa gctccaaaag gcgaattagg aatttatctg 1920
gtaggagatg atagtctttt cccctggaga tggaaaattc gtccaccggg ttttattaat 1980
ttgcaaattc ttcctcatct agttaaaaaa atgaaattgg ctgatatcat gacaatatta 2040
ggtagtatag atatcattat gggggaagtt gatcgttgaa atgataatag atagggtaga 2100
ggtagaaact atcaattctt tttcgaaatc agaattattt aaagaaatct acgaacttat 2160
atggattcta cccatttttg ccctcctact gggaatcaca atagaagtac tcgtaattgt 2220
gtggttagaa agagaaatat ccgcatcgat acaacaacgt attggtcctg aatatgctgg 2280
ccccctggga ctgcttcaag ctatagcaga tggaactaaa ctacttttaa aagaggatat 2340
cctcccatcc cgaggagata ttcctttatt tagcattggt ccctctatag cagtcatatc 2400
cattttatta agttttttag ttatcccttt aggatatcgt tttgttttag ctgatcttag 2460
tattggtgtt ttttatggat tgcgatttca agtattgctc ctattggtct tctcatggca 2520
ggatatagct caaataataa atattctttt tcaggcggtc tacgagcggc tgctcaatct 2580
attagttatg aaataccatt aactttttgt gtactagcaa tatctctacg tgtgattcgt 2640
taaaatagga tctttttcct ctaaaataca ttgaatgctt atcttccttt gcttattctg 2700
tattcgcgtt ggtaagttaa actcgatagc tatatgagtg aaacaaaaca gcttattaat 2760
ttgtagtaaa agtaaaaaat ctcatttcct acgtacaaga aaaaagttca agtaaacata 2820
agcagtgtaa actcttaacc ccaaggttga gattgtttga ttagtcatca tatcttgaag 2880
cgggcaagaa taaaagattc gcgatatgga attccattac tagaatattt tgagttatta 2940
ctataattta aacttataac cacaaggcaa tcgatcaaaa tttagtgagg gattaggaac 3000
actaaagtac atacaagatt agtaatgaga gaatctaaat cattagacat ttttcgtcat 3060
aaaaggaatc ataataagga cttgaaattg gtggaaatga tcaagccgta ctttcttcag 3120
attccggtct agagtatgtt cccattcact tgttaaggaa atggctatca agaacgaatt 3180
aaccctttat tcttttttta agtatacccc tcctagggaa agaagagtag gacaaaagat 3240
aaggaataca atacaaaaaa gatctttatt tattctttcc ttcctttatc cctattcata 3300
cagaattcct catgaactaa tgccaaattc tttccattta ttaattgcta caacgagtga 3360
tttattccaa tattaagtta ttaccgaaca aagcaaaatt atattatata aaggatgaga 3420
tcaattcgga agcgcttttt tgttattcta gcagacgtaa ttgctttggt ctaattttgg 3480
gctttccaat caattttatc ttatctaatt ctatctatgc ccagaggatg ataccgaaac 3540
gaaacaatcc tttccttttt tctgatcata gaggagccgt atgaagctaa ggtttcatgt 3600
acggttttgg aatagcggtg agaactgtga tgttatcatc gactatgatt atctaatagt 3660
tcaagtacag ttgatatagt tgaagcacag tccaaatatg gtttttttgg atggaatatt 3720
tggcgtcagc ctataggttt tctagttttt ctaatttctt ctttggcaga atgtgaaaga 3780
ttaccctttg atttaccaga agcagaagaa gaattagtag caggttatca aaccgaatat 3840
tctggtatta aatatggttt attttatctt gtttcttacc taaatttatt agtttcctct 3900
ttatttgtaa ctgttctata cttaggcggg tggaatttct ctattcccta tatatccttt 3960
tttggatttt tccaaatgaa taaaataatt ggaattttgg aaatggtaat aggtatcttt 4020
attacattaa ctaaagctta tttatttctc ttcatttcta tcacaataag atggacttta 4080
cccataatga gaatggatca gttattaaat cttggatgga aatttctttt acctatttct 4140
ctgggcaatc tcttattaac aacttcttcc caactagttt cactataaat aagaatacaa 4200
taacagtaag aatattttca acacaaacgt tctctcaaac aagagaaaga aacatacctt 4260
tttcatatat agatttagaa tatgttccct atgctaactg ggttcattac ttatggtcaa 4320
caaacaatac gcgccgcaag atacattggt caaagtttca taattacctt atcccacaca 4380
aatcgtttac ctataacgat tcactaccct tatgaaaaat caattacatt ggagcgtttc 4440
ctggggcgaa tacactttga atttgataaa tgcattgctt gtgaagtatg tgttcgcgta 4500
tgcccgatag atctaccctt tgtggattgg agatttgaaa aggatattaa aagaaaacaa 4560
ttgcttaatt atagtattga tttcggagtt tgtatatttt gcggtaactg tgttgaatac 4620
tgtcccacaa gttgtttatc aacgactgaa gaatatgaac tttctactta tgatcgtcat 4680
gaattgaatt acaatcagat tgctttgagt cggttaccat aatgggagat tacacaattc 4740
aaacaattag aaatttgcct caaagtaaaa tagacgaaga aaaatcttgg aattcaagaa 4800
cgattacaga ttactaggta ttaggatttt ttttattaga aaaatccatt tttactaact 4860
ctaacgaaaa agaataacta ctgattaaca acttatatgt atatacaaaa aaatatccta 4920
ataccttttt ccttccttga atcttttagt tttagtcagt tcatgaaaaa ttttatacta 4980
gaaatttctt cttatccata atggatttac ctggaccaat acatgagatt cttgtgctat 5040
ttgggggatt tggtcttcta ctaggaggtc taggagtagt attacttacc aacccaattt 5100
attctgcctt ttcgctggga ttagttcttg tttgtatatc cttattctat tttttattaa 5160
attcctactt tgtagctatc gcacaacttc ttatttatgt gggagccata aatgtcttga 5220
tcatatttgt tgtaatgttt gtaaacggct cagagtggtc taaagataag aattattgga 5280
ctattggaga tgggtttact ttactccttt gtataactat tcctttttca ctaatgacta 5340
ctatcccaga tacgtcgtgg catggaattc tttggactac aagatcaaac caaatagtag 5400
aacagggtct cataaataac gttcaacaaa ttgggattca tttagcaacc gatttttatc 5460
ttccatttga actcatttcc ctaattcttg tagtttcttt aataggtaat tactatggct 5520
cgacaataag aaatacttag aatgagtcaa aattcttaga atttcaaata taaaataaat 5580
aactaaagaa tcacaatttt gatttagtaa aacccatcta ctgccaatac aacaaatacc 5640
ttcttttctc ttttgttgcg taattgttct attctagtta attgaatcag ttcaattctt 5700
gtcctcatat tgaaatgaat cgagattgat aaggaggtag ttaatgatgt ttgagcatgt 5760
actttttttg agtgtctatt tattttcgat tggtatctat ggattgatca caagccaaaa 5820
catggttaga gctctaatat gtcttgaact tatactaaat tcaattaatc taaatctcgt 5880
aacattttct gatctatttg atagtcgcca attaaaagga gacattttcg caattttgtt 5940
atagcccttg cggctgctga agcagctatt ggactatcca ttctttcttc catccatcgt 6000
aacaggaaat caactcgtat caatcaatcc aattttttga ataattagac atagaatccc 6060
ctaaacaaag gggcatatat aataataaga aacattaaga tgaatagaaa tctaatctta 6120
ttttcttatt agtgtttaat aatatccttt ttttgagtag gttatttcag agtattgttt 6180
tttacttatt gaatattgca tttttgcaat tcattgatat tgcaatttga atattgcaat 6240
aatttatatt gaaaagatga tagccaattt attggctaat tcgaattagt atgtagaatt 6300
tgtataatta taactgttga agccttaaat tcaagtctct tggctctttt cacgctttct 6360
cacaaacaga ttacgaaata tattgcatta tttgttaaag tttggataaa ctattgcttc 6420
gtctggtgtc tacaatacat ctaatttata tagtactaat ttcattttta ccagatcgaa 6480
aatttttatg ttgaaaagga aaatttagag atccaatgtc acattctgta aaaatttatg 6540
atacatgtat aggatgcact caatgtgtac gagcttgccc aacagatgta ttagaaatga 6600
taccttggga tggatgtaaa gccaagcaaa ttgcttccgc gccgagaacc gaagattgtg 6660
tgggttgtaa gagatgcgaa tccgcctgtc caacggattt ttaagtgtcc gcttttattt 6720
agggcctgaa acaacccgca gcatggctct atcttattga tacgttacaa aaaaactcca 6780
cttgaatcgt ctgattcctc tttaccgaag aagcctgtgc tcgaaataat cgagcatggg 6840
cttttctgat caaaacgtat cttgtattta ttactttatc atgagttatt ttccttggtt 6900
aacaatactt gttgttttgc cgatatttgc aggttcatta attttctttt tacctcataa 6960
aggaaataaa atcatatact atagctattt gtttattaga attccttcta atgacttatg 7020
cattctgtta tcatttccaa ttggaggatc ctttaatcca attaaaggag gattctaaat 7080
ggatagatgt tttcgatttc cactggagat tgggaatcga tggactttca ttaggatcta 7140
ttttattgac aagatttatc actactttag ctactttagc ggcttggccg gttactcgga 7200
attcgcaatt attctatttc ctgatgctag caatgtatag tggtcaaata ggattatttt 7260
cttcatgaga ccttttactt ttttatcatg tgggagttag aattaattcc tgtttactta 7320
cttttatcca tgtggaggga aagaggcgta tgtattcagc taccaagttt attttgtata 7380
ctgcaggcgg ttccattttt ttcttaattg gagttctggg tatgggatta tatggttcca 7440
atgaacccgg attagattta gaaagattga ttaatcaatc ataccctaca acattggaaa 7500
tactactgta ttttggcttc cttattgctt atgctgtcaa attgccgatt atacctttac 7560
atacgtggtt accagatacc catggggaag cgcattacag tacatgtatg cttttagccg 7620
gaattctatt aaagatggga gcatacggat tgattcgggt caatatggaa ttgttaccgc 7680
atgctcatta tctattttcc ccttggttgg taataatagg agcggtgcaa ataatctatg 7740
cagcttcaac ttctcttggt caacgaaatt tcaaaaaaag aatagcctac tcctccgtat 7800
ctcacatggg tttcataatt ataggaattg gttccataac caacattgga ctaaatggag 7860
ctattttaca aatattatct catggattta tcggtgctac actttttttc ttggcgggaa 7920
cggacttgtg atagaatgcg tcttgtttat ctcgaagaac tggagggaat atctatccca 7980
atgccaaaaa tttttaccat gtttagtagc ttttcagtgg cttctcttgc cttgccggga 8040
atgagcggtt ttgttgcaga attagtagta ttttttggac taattactag tcctaaattt 8100
atgttaatgc caaaaatgct aattactttt gtaatggcaa taggaatgat attaactcct 8160
atttatttat tatctatgtt acgccagatg ttctatggat acaagctatt tcatgttcca 8220
atcaaaaatt ttgtagattc tggaccacgg gaactctttc ttttaatctg tatcttttta 8280
ccagtaatag atctatctcg aattttcaga attagatcta tctcgaattt ttcagaatta 8340
gatctttttc gtatctttca gaattagcta tttcatgttc catctttttc agaattagat 8400
ctatctcgaa tttttgagaa ccccttgaac gtcttttcaa agggttctca aatcaaacta 8460
aaaaggaaaa aaaccgaagg ttttatgtta tgtaattatt tagatggtaa tgtaaatgaa 8520
ccgtaactat gtaaacctat tcctaacaga ttgataccaa aatagcagat ccaaattata 8580
agaaatccta tcgaagctac aagtgcggaa ttcgtaccct tccaatttgg attttttcta 8640
ctatgtaaat atattgcaaa tatggtccag gtaataaatg cccaagtttc cttaggatcc 8700
caattccaat aggatcccca tgcctcatta gcccatactg ctccacaaag aatacccacg 8760
gttaaaagag taaaccctag actaatgaca cgataactcc aagaatccaa acgctcagtt 8820
aattgatatt tgtaataatt tggaaatacg ggaaaagagg tgttttttaa agcacttctt 8880
tttgcatata aatattcaat ctcactaaag aaaaatgttt taagaaaaac atttttcttt 8940
agtgaaaaga aatcgaaatt ctttcgaaat ctaatgatta gaagagcggc ggataataag 9000
gatccacaca aaagagttgc atagcttagt aacatcatac tgacatgcat cattaaccac 9060
tgagattgta gagcaggtac tagtattgtg gattgatgca tttcagttaa aagacccgac 9120
gtggcaaagc cttgcgttaa aatagtactt ggcgtagtta ttgtgcttaa atcatttttc 9180
gagttctgta tcttaggaat agtatgaaga atatacagag tccatgaaag gaagatcaat 9240
gactcatata aattacttaa tggaaaatgt cccgaagaaa cccaacgaga gactaaaaat 9300
cctgttatag agaaaaaagt agctatcatt cctttttctg acgaatcacg taatccccta 9360
agttcacgaa ctaataaggt tatcaaatga atcgtaatca caattgaaat ggttgagaaa 9420
gagatatgag ttagtatatg ttctaaagtt gcaaatagca taacgataag gtcccattac 9480
aaaattggaa atttcgaatt gaatccattt tctaattttt gtattctttt tcgagaatgc 9540
cgccactcgg attcgaaccg agatgcttga gcactgcttc ctaagagcag cgtgtctacc 9600
aatttcacca tggcggctaa tttaaaataa tagttaactt aaaagaataa tagttatttt 9660
atcgtgaatc gtcgagactg ggagaagcca tagaatttag gaacattaga agttcatcat 9720
tagaagttca tcattaacta caatgaaatg cattttgtat tttagaaaaa tgatagaatg 9780
aaagccttta cactcttatt aatatgatgt accagtccta aacccattta tatgggaatt 9840
ttggataaga ttaggtggag gttgtaagtc ctattgcaag aatagttact ttttataata 9900
gaatcctcgt ttttatgaga attctattat aaaaaaaagt tctttgatag gaaaagaata 9960
ctgaggacac aatataccaa aaacttttgt tctatataat gataatggag ggattcgttc 10020
taagaatatt gtaccgagga attcgacaca taaaagtaca tttattaatt aatccgaatt 10080
attcattcat attaaataat tggaatttct tttggatagt tcaattcaga ttatttcaaa 10140
atcttattat ttgtttgctt gttgcccaga aaaacctttt ggttttggat gctcgttgcc 10200
cctagaaaat gatcttgatt ttgctaaaga ataagattgt actatggaaa aataagtctt 10260
ttttttccaa agatttttac gaatacgctt ttttgacatc gaagtacgtt tttttggaac 10320
tgccattcaa aaagggaatt acttttttct agttgtatgt gaaagacatc tattgccaca 10380
aatcaatcct tctttctgtt ttttcttagt atttatactt agatactgaa agatacttaa 10440
attctaaatt cttctttagt tcattttgtc taatgtggat aagacaagag ttttttaaga 10500
tttttattta aatcaatttc tatatcaaat atactccata tataaatata tggtatgggg 10560
gataagccct catataagag ggggagataa aaagaaggta aaattcaatt caaatagtta 10620
attaagttca gaaggctatt ccataattga attccaataa aaaaaaatac ttagtctctt 10680
aaacaagaca ttctaaaact tagagaattc tagtcattag gatttccttt tatatgaaat 10740
aagcaatatt cgagaatttc ctataaatat aggttttctt tggaattcaa tcaatcaatt 10800
acgaaacaag agagctcttt tattttaaca aaaagaaata caaaaatgat tagaaagtaa 10860
aattattcaa tctttttttg tattttaata aatatttttt tttactaata actagatacc 10920
gaaattcttt gattcacttt ttgaatttaa gtaactaaac ccattctaaa ttttggaata 10980
ttttaatgag agaaattaga aaaattcata attccagtat tttttatttt tcttattttg 11040
tttttttaat tcctttagaa agagataaga ataggtttgg tgaatcggaa acaattttat 11100
tttatagaaa aattgaacta taaatttaaa ctaaaaattg ctatttcttt tcttatggaa 11160
catacatatc aatatgcctg ggtaattcct cttctcccac ttccagttat tatgtcaatg 11220
ggatttggac tttttcttat tcctacagca acaaaaaatc ttcgtcggat atgggctttt 11280
cctagtattt tactcttaag tatagctatg gtattctcac ttcacctgtc tattcaacaa 11340
ataaatggaa gttctatcta tcaatatcta tggtcttgga ccatcaataa tgatttttcc 11400
ttagaatttg gatacttggt cgaccccctt acgtctatta tgttaatact aattactact 11460
gtaggaatct tagttcttat ttatagtgac gattatatgt ctcacgatga aggatatttg 11520
agattttttg tttatataag tttttttaat acttccatgt taggattggt tactagttcc 11580
aatttgatac aaatttattt tttttgggaa cttgtcggaa tgtgttccta tttattgata 11640
ggcttttggt ttacgcggcc aattgcagcg agtgcttgtc aaaaagcttt tgtaactaat 11700
cgtgtagggg attttggtct gttattagga attttaggtt ttttttggat aacgggtagt 11760
ttggagtttc gggatttgtt caaaatagct aataactgga ttcctaataa tgggattaat 11820
tccttactta ctactttgtg tgctttttta ttattccttg gtgcagttgc aaaatctgca 11880
caatttcctc ttcacgtatg gttacctgat gctatggaag gacccactcc tatttcggct 11940
cttatacacg cagcaactat ggttgctgcg gggatttttc ttctagctag acttcttcct 12000
cttttcatat ccctaccctg gataatgagt ttcatttctt taataggtac aataacactc 12060
ttcttaggag ccactttagc tcttgctcag agagatatta aaagaagctt agcctattct 12120
acaatgtctc aattgggtta tatgatgtta gctctaggta taggttctta tcaagctgct 12180
ttattccatt tgatcactca tgcttattcg aaagctttat tgttcttagg atccggatcc 12240
gttattcatt caatggaacc tcttgttgga tattcaccag ataaaagtca gaatatggtt 12300
cttatgggtg gtttaagaaa atacgttcca attacaagaa ctactttttt atgtggtaca 12360
ctttctcttt gtggtattcc acctcttgct tgcttctggt ccaaagatga aatccttagt 12420
aatagttggt tgtattcacc cttttttgga ataatagcct cttttactgc aggattaact 12480
gcattttata tgtttcggat atatttactt acttttgatg ggtatttgcg tgttcatttt 12540
caaaattaca gtagtactaa agaaggttcg ttgtattcaa tatccttatg gggaaaaagt 12600
atatccaaag gagtcaatag ggattttgtt ttatcaacaa tgaagagtgg agtttctttt 12660
ttttcacaaa atataccaaa aattcctgct aatacaagaa ataagatagg atcctttagt 12720
actccctttg gggctaaaaa tacttttgtc tatcctcatg aaacgggaaa tactatgcta 12780
tttcctcttc ttatattact actttttact ttgttcattg gatccatagg aatccatttt 12840
gataatggag taaaagataa tagaatattg gagttaacca tattatcaaa gtggctaact 12900
ccttcaataa acttgttcca ggaaaattct aattcttcca taaattcata tgaatttctc 12960
actaatgcaa tttcttctgt aagtttagca atttttggtc tattcatagc atatatcttt 13020
tatggatctg cttattcttt ttttcagaat ttgaattttc aaaattccct tgtaaaaaag 13080
aatccaaaaa agagcttttt ggatgaagta aaaaaaaaga tatacagctg gtcatataat 13140
cgtggttata tagatttttt ctatactagg gtttttatcc taggtataag aagattagcc 13200
gaactaacgc atttttttga taaaggtgtc attgatggaa ttaccaatgg agtaggtctt 13260
gctggttttt gtataggaga agaaatcaaa tatgtagggg gagggcgaat atcgtcttat 13320
ctattctttt ttttatgtta tgtatccttg ttcttattct ttattccatg aaaatggatt 13380
attccatgaa ttcctcaaaa cgaggctcat caaaatgcaa aatctaagac tactataaga 13440
ctactaataa aataagaaaa aaatgagaac gattaaatta cttctcccga atatccaact 13500
gacttattaa tttcttataa cgtactctat ttttctttgc caaataagcc agcaaacgtt 13560
gacgttttcc caaaagtctt cgtagacctc tttccgatga aaaatctttt ttgtgtaatt 13620
ctaaatgtga agcaagtctc cgtatcttat tggtgaaact gaatacttga aattcaacag 13680
aacccctgtt ttcttgtttt tcttctttaa ccataaatca acaaattttt ctacctcctt 13740
tctttttcat gtatttttct gatcaggaaa aataaaaaat tatgtcagtt atttttaagt 13800
tattctaatc tcgtacacac aaaaatttgc aattattcat ctactgctgg aatctggaat 13860
ttggatttat tttatcgatg caaattggat ttggatagaa gggtacattc ttttatttta 13920
gatagaagaa acatttcttc tatctaaaat aaaagaattt tgctgattta tttattgcta 13980
tatccaattt atagaattga tataccattt aattgatatc atttagcaaa atgaaacaca 14040
gcatatgcat ccatctttgg gctcgagaat ttcacgggat agagatatag tataagaaat 14100
aggctattaa gtaactctaa atgaattgtg gatacatctg tatccttaac atactgaaac 14160
gactgccatt attcgtatca aaccaatagc gattcataaa agctaaatct tgtaatcaat 14220
ggtgggccaa taatgaattt ttttgcatat gtattaagac gcttggtctg atttcgaaat 14280
tgtccagagt tttttatgtt gttttcattg caaaatgatg gatctccttc cgtaactttc 14340
caattacgag tacgagaatt gaaagacatg aaaattctca attctctacg gcgtctagga 14400
gatggatcga atattttcag gaacaagaaa atcagaagaa tctttttctc tattcactac 14460
cattccgcgt cttcgacttc tattagtttc ttttcttctt taatgcaata gctatagttt 14520
gatatagaat ccatttctca aagtaatgga aaccattctc ttataggaaa tggttcgaaa 14580
atcgctattc caccttttag gtttaggtat cgtgaaaagt gatacctgtg aagatcgtgc 14640
atttcagtca cattcagatc cgtttttcga gtccatgata taaccaaatt ggatggatct 14700
tccacccgtt tagctaagaa agaatagatg cagaggtgga taatagatcg atatgaagat 14760
catgagctgc cccataatga aaccgccagt agtcgcgaat atctccttct tccctaaccc 14820
aagattggag aaagaagatc taagagggac ctatggagaa tgtggtcaga aatccataat 14880
agagtccgac cacaacgacc gaattaatta tcctcatcga gaaacttaga ctactttaga 14940
ctacttaagt agaaaagatt tgaaaatcat ggcatgggtc tccttttttt ctttctttag 15000
agttttctat atgcacaatt tctcgatgtt tcgatgagaa tttcttgact ttccatatat 15060
agaaagagat agactataaa tgacatctct tatgtcaata agaccaaagg aatggatatt 15120
aaatgatagg aagtgctaag aagtgaaata gaatgaaata gagccacttt gggcttccct 15180
atgaaatgag gcatggaacg gagccactac gaagaaattc cgggagttac gaaagaagct 15240
tcggactcat attgttcacg ggttgagagc gggagttgaa ctctaggagg tcgaatctcc 15300
ccttgttcct cagtagctca gtggtagagc ggtcggctgt taactgactg gtcgtaggtt 15360
cgaatcctac ttggggagat ttgattcatt ctttaatgta agaataaaga attgaattaa 15420
aaggcttgct ttgaccctta ggagtaggta acccgttcgc tatccttgtt tctattgcat 15480
tctgtctcat cgtatcacat tctgttctac gattccactt cgacaaaagg aaagagcata 15540
cccaagttca atagctttac gtccgctatt ccgatcatga ttttcctacc ctcagggaga 15600
aagtaaaggt ccttccccct ttggaaggct gtgggcgagg agggattcga acccccgaca 15660
ccgtggttcg tagccacgtg ctctaatcct ctgagctaca ggcccacccc gtctccactg 15720
gatctcttcc cggggatacc ccccaaaagg aaccttcttc tcctcagcca tttcatttcg 15780
ggttaagaag atgggaaagc gcctttctct ctataagaac agtgcgttct gaggtgtgaa 15840
gtgggagaga ggggatgatt gaggttttga ataagacgac ctttgcgttt tggatttgga 15900
tctttttcgt atttcaaaat agtgaaaaag tcaaataaga ggtgttaagc tttttatcat 15960
tctggcatcg agctattttg ccgcaggacc tcccctacag tatcgtcacc gcagtagagt 16020
ttaaccacca aattcgggat ggattggtgt ggttcctcta cgcctaggac accagaatat 16080
cgaaccatga acgagaaaag gcatgagaga aatattggct agtaattgtg aagtcccaat 16140
tcttaactgg aagggacacc aaaggactct gccctccctc tctatttatc caagagatgg 16200
aagggcagag cttttttttg gttttttcat cttttctttt catcaaagag ttgaacaatg 16260
aagatagatg gcaagtgcct gatcgatttg atcaggtcgt gtaggaacaa ggttcaaatc 16320
gttcgttcgt taggatgcct cagctgcata catcactgca cttccacttg acacctattt 16380
aaacggctcg tctcgccgct accttatcct atttccatac ttctgtcgct ccatccccgt 16440
atgggtggag aacccgtcgc tgtctcggct gtgctaccgg aggctctagg gaagtcggag 16500
gagagagcac tcatcttggg gtgggcttac tacttatatg ctttcagcag ttatcctctc 16560
cacacttggc tacccagcgt ttaccgtagg cacgataact ggtacaccag aggtgcgtcc 16620
ttcccggtcc tctcgtacta gggaaaggtc ctctcaatgc tctaacgccc acaccggata 16680
tggaccgaac tgtctcacga cgttctgaac ccagctcacg taccgcatta atgggcgaac 16740
agcccaaccc ttggaaccac ctacagctcc aggtggcgaa gagccgacat cgaggtgcca 16800
aaccttcccg tcgatgtgga ctcttgggga agatcagcct gttatcccta gagtaacttt 16860
tatccgttga gcgacggccc ttccactcgg caccgtcgga tcactaaggc cgactttcgt 16920
ctctgctcga cgggtgagtc ttgcagtcaa gctcccttct gcctttgcac tcgaggacca 16980
atgtccgtct ggcccgagga aacctttgca cgcctccgtt accttttggg aggcctacgc 17040
cccatagaaa ctgtctacct gagactgtcc cttggcccgc gggtctgaca caaggttaga 17100
atccgagctc ttccagagtg gtatctcact gatggctcgg gcccccccgg aagggggcct 17160
tcttcgcctt ccacctaagc tgcgcaggaa aagcccaaag ccaatcccag ggaacagtaa 17220
agcttcatag ggtctttctg tccaggtgca ggtagtccgc atcttcacag acatgtctat 17280
ttcaccgagc ctctctccga gacagtgccc agatcgttac gcctttcgtg cgggtcggaa 17340
cttacccgac aaggaatttc gctaccttag gaccgttata gttacggccg ccgttcaccg 17400
gggcttcggt cgccggcttc cctgtcatca gttcaccaac ttccttgacc ttccggcact 17460
gggcaggcgt cagcccccat acatggtctt acgactttgc ggagacctgt gtttttggta 17520
aacagtcgcc cgggcctggt cactgcgacc cccttttgcg agggggcacc ccttctcccg 17580
aagttacggg gctattttgc cgagttcctt agagagagtt gtctcgcgcc cctaggtatt 17640
ctctacctac ccacctgtgt cggtttcggg tacaggtacc cttttgttga aggtcgttcg 17700
agcttttcct gggagtatgg catgggttac atacttcagc gccgtagcgc ctggtatgag 17760
cctcgtggag aagcaatggc tagtccacgg ggctcatact tcagcgctgc agcgcttggt 17820
actcggacct cggctcgagg cattttctct accccttctt accctgaaaa agcagggtca 17880
ccttgtgtcc ttaaacctat aaccatcttt cggctaacct agcctcctcc gtccctccgt 17940
accaacaagg ggtagtacag gaatattgac ctgttgtcca tcgactacgc ctttcggcct 18000
gatcttaggc cctgactcac cctccgtgga cgaaccttgc ggaggaaacc ttgggttttc 18060
ggggcattgg attctcacca atgttttcgt tactcaagcc gacattctcg cttccgcttc 18120
gtcgaccccc gctttcgcgt ttgcttccct ctaaggcgga acgctcccct accgatgcat 18180
tttgacatcc cacagcttcg gcagatcgct tagccccgtt catcttcagc gcaagggcgc 18240
tcgatcagtg agctattacg cactctttaa agggtggctg cttctaggca aacctcctgg 18300
ctgtctttgc acccccacct cctttatcac tgagcggtca tttaggggcc ttagctggtg 18360
atccgggctg tttccctctc gacgatgaag cttatccccc atcgtctcac tggccgacct 18420
tgacccctgt tatttttggg tcatatctag tattcagagt ttgcctcgat ttggtaccgc 18480
tcgcgcagcc cgcaccgaaa cagtgcttta cccctagatg tccagtcaac tgctgcgcct 18540
caacgcattt cggggagaac cagctagctc tgggttcgag tggcatttca cccctaacca 18600
caactcatcc gctgattctt caacatcagt cggttcggac ctctgcttag tttcatccaa 18660
gcttcatcct ggtcatggat agatcaccca ggttcgggtc cataagcagt gacaatcgcc 18720
ctatgaagac tcgctttcgc tacggctccg gtgggttcca ttcccttaac caagccactg 18780
cctatgagtc gccggctcat tcttcaacag gcacgcggtc agagatcact ttcccctccc 18840
actgcttggg agctcagcac ggtttcacgt tctatttcac tacccactgg gggttctttt 18900
cacctttccc tcacggtact acttcgctat cggtcaccca ggagtattta gccttgcaag 18960
gtggtccttg ctgattcaca cgggattcca cgtgccccat gctactcggg tcagagcgta 19020
agctagtgat gctttcggct actggacttt agccatctag ggtgcggcac tcaaccgctt 19080
cgcctagcag cacaacgctt gtattgctct cccacaaccc cgttttcacg gtttaggctg 19140
ctcccatttc gctcgccgct actacgggaa tcgcttttgc tttcttttcc tctggctact 19200
aagatgtttc agttcgccag gttgtctctt gcctgctcat ggattcagca ggcagttcaa 19260
aaggttgacc tatttgggaa tctccggatc tatgcttatt ttcaactccc cgaagcattt 19320
cgtcgcttgc tacgcccttc ctcgtctctg ggtgcctagg tatccaccgc aagcctttcc 19380
tcttttgaac ctcgccatta acgttaaggc tatgccatcc taaggtgcta ctaaatggaa 19440
ggatcttatc aacgtccatg aatgcgaaat catagatcga actgacgaat tggcaacctt 19500
cggtgctatc atagtatccg ctaagttcac gggctggaga taagcggact cgaaccgctg 19560
acatccgcca cagggtaaac caccgcctct caggcctccc cgacgggttc taccatagag 19620
gacaacgata ggcaataact cccccccgaa cacagcttac aactttcatc gtactgtgct 19680
ctccaaagag caactcttct caaaatccca aaacaaaagg tgctgagttg gaatcccatt 19740
ctaaggattc ttgtggttcc ggggaatcca gctacaggag aaccaggaac ggggagctct 19800
cccctttttc cgcccgactc tttgatctta acttaagaat gctggtttta agaacgagtg 19860
attgcccttc tccgaccctt actgcccaac cggagagcgg acggctaatg tgttccactt 19920
attgaacagg atctatggtc ggtccgtgac ccctggacgc cgaaggcgtc cttggggtga 19980
tctcgtagtt cctacggggt ggagacaatg gggtcggtcc atggattttc cttccttttg 20040
ccacatttcg ctcaaagggt tgaagggaga tagtgcatca agctattcgc aagggccaac 20100
ttgatcctct tccccaggga tcccagatga gggaagccta ggagagccgc cgactccaac 20160
tatcgtccat gtacgatcca tactagatct gaccaactgc ccatcctacc tcctctacct 20220
ttttgacagc ccatcttttt gtctcagtag agtctttcag tggcatgttt cagtcctctt 20280
ccccattact tagaaaaagt gagccaccgg ttcaggtaca agatactatc attaccgcct 20340
ggacaattag acagccaacc cgtaatcgca acgacccaat tgcaagagcg gagctctacc 20400
aactgagcta tatccccccc gagccaagtg gagtatgcat gaaagagtca gatgcttctt 20460
ctattctttt ccctggcgca gctgggccat cctggacttg aaccagagac ctcgcccgtg 20520
aagtaaatca tcgcccctac gatccaacca attgggagag aatcaataga ctccttttcg 20580
ggagcgattc atccttcccg aacgcagcat acaactcccc gttgtactgc gctcttcaag 20640
tgtgcttctt cccccttctc ccccttacca cggcaagtcc ttgggaaata actccgatgg 20700
gcagaaaaaa gaaggggtta agagaccctc ctggcccaac cctagacact ctaagatcct 20760
ttttcaaacc tgctctgctc ccatttcgag tcaagagata gataaatagc cacatcccat 20820
tgcactgatc gggggcgctc gtagtgactt agggggtcga agaccaagaa gtggcttatt 20880
tataccaagc attcctctta tggctagatc caatctcctg gtccctgcag aaaggaaaaa 20940
gaatttcacg ttcttccttt caggaaggga ggattaggga agtcctattg attactgctt 21000
tctccagacc gccgggaaaa gcatgaaaaa aaggctcgaa tggtacgatc cctccgtcac 21060
cccagaatga aaggggtgat ctcgtagttc ttggtctgtg aagatgcgtt gttaggtgct 21120
ccattttccc attgaggacg aacctcaacc tgtgctcgag agatagctct ccatacactg 21180
ataagggatg tatggattct cgagaagaga ggagccgcgg tggccccccc ccggaccgcc 21240
cggatcccac gagtgaatag aaagttcgat ctacatggga tctcacctga atcgccccat 21300
ctatcctcct gaggagaagt ttgtttggtt tcaaactccg attcaaacag gaggagtacg 21360
ccatgctaat gtgccttgga tgatccacat cttcgggtca ggcgctgatg agcacattga 21420
actatccatg tggctgagag ccctcacagc ccaggcacaa cgacgcaatt atcaggggcg 21480
cgctctacca ctgagctaat agcccgtcgc gcgggcctcc caaagggagg cctgctacgc 21540
caaaagcgag aaaaactcca tccctttcct tttgacatcc ccatgccgcc acacgggggg 21600
acatggggac gtcaaaaagg ggatcctatc actatcaact aatttgttcc gacctaggat 21660
aataagctca tgagcttggt cttacttcac cctaaacgaa agaagacttc catatccaag 21720
tttagctcag acgtagctgc cttctttttg ggcgtgaagc agtgtcaaac caaaataccc 21780
aataagcata agcattagct ctccctgaaa aggaggtgat ccagccgcac cttccagtac 21840
ggctaccttg ttacgacttc actccagtcg caagcctagc cttaggcatc cccctcctta 21900
cggttaaggg taatgacttc aaacctggcc agctcctata gtgtgacggg cggtgtgtac 21960
aaggcccggg aacggattca ccgccgtatg gctgaccggc gattactagc gattcctgct 22020
tcatgcaggc gagttgcagc ctgcaatccg aactgaggac gggtttttgg agttagctca 22080
ccctcgcgag atcgcgaccc tttgtcccgc ccattgtagc acgtgtgtcg cccagggcat 22140
aaggggcatg atgacttggc ctcatcctct ccttcctccg gcttaacacc ggcggtctgt 22200
tcagggttcc aaactcatag tggcaactaa acacgagggt tgcgctcgtt gcgagactta 22260
acccaacacc ttacggcacg agctgacgac agccatgcac cacctgtgtc cgcgttcccg 22320
agggcacccc tctctttcaa gaggattcgc ggcatgtcaa gccctggtaa ggttcttcgc 22380
tttgcatcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca attcctttga 22440
gtttcattct tgcgaacgta ctccccaggc gggatactta acgcgttagc tacagcactg 22500
cacgggtcga gtcgcacagc acctagtatc catcgtttac ggctaggact actggggtct 22560
ctaatcccat ttgctcccct agctttcgtc tctcagtgtc agtgtcggcc cagcagagtg 22620
ctttcgccgt tggtgttctt tccgatctca atgcatttca ccgctccacc ggaaattccc 22680
tctgccccta ccgtactcca gcttggtagt ttccaccgcc tgtccagggt tgagccctgg 22740
gatttgacgg cggacttgaa aagccaccta cagacgcttt acgcccaatc attccggata 22800
acgcttgcat cctctgtctt accgcggctg ctggcacaga gttagccgat gcttattcct 22860
cagataccgt cattgtttct tctccgagaa aagaagttga cg 22902
<210> 25
<211> 1029
<212> DNA
<213> maize
<400> 25
atggatttac ctggaccaat acatgagatt cttgtgctat ttgggggatt tggtcttcta 60
ctaggaggtc taggagtagt attacttacc aacccaattt attctgcctt ttcgctggga 120
ttagttcttg tttgtatatc cttattctat tttttattaa attcctactt tgtagctatc 180
gcacaacttc ttatttatgt gggagccata aatgtcttga tcatatttgt tgtaatgttt 240
gtaaacggct cagagtggtc taaagataag aattattgga ctattggaga tgggtttact 300
ttactccttt gtataactat tcctttttca ctaatgacta ctatcccaga tacgtcgtgg 360
catggaattc tttggactac aagatcaaac caaatagtag aacagggtct cataaataac 420
gttcaacaaa ttgggattca tttagcaacc gatttttatc ttccatttga actcatttcc 480
ctaattcttg tagtttcttt aataggaatc cattttgata atggagtaaa agataataga 540
atattggagt taaccatatt atcaaagtgg ctaactcctt caataaactt gttccaggaa 600
aattctaatt cttccataaa ttcatatgaa tttctcacta atgcaatttc ttcttcaagc 660
tcccttctgc ctttgcactc gaggaccaat gtccgtctgg cccgaggaaa cctttgcacg 720
cctccgttac cttttgggag gcctacgccc catagaaact gtctacctga gactgtccct 780
tggcccgcgg gtctgacaca aggtaccctt ttgttgaagg tcgttcgagc ttttcctggg 840
agtatggcat gggttacata cttcagcgcc gtagcgcctg gtatgagcct cgtggagaag 900
caatggctag tccacggggc tcatacttca gcgctgcagc gcttggtact cggacctcgg 960
ctcgaggcat tttctctacc ccttcttacc ctgaaaaagc agggtcacct tgtgtcctta 1020
aacctataa 1029
<210> 26
<211> 342
<212> PRT
<213> maize
<400> 26
Met Asp Leu Pro Gly Pro Ile His Glu Ile Leu Val Leu Phe Gly Gly
1 5 10 15
Phe Gly Leu Leu Leu Gly Gly Leu Gly Val Val Leu Leu Thr Asn Pro
20 25 30
Ile Tyr Ser Ala Phe Ser Leu Gly Leu Val Leu Val Cys Ile Ser Leu
35 40 45
Phe Tyr Phe Leu Leu Asn Ser Tyr Phe Val Ala Ile Ala Gln Leu Leu
50 55 60
Ile Tyr Val Gly Ala Ile Asn Val Leu Ile Ile Phe Val Val Met Phe
65 70 75 80
Val Asn Gly Ser Glu Trp Ser Lys Asp Lys Asn Tyr Trp Thr Ile Gly
85 90 95
Asp Gly Phe Thr Leu Leu Leu Cys Ile Thr Ile Pro Phe Ser Leu Met
100 105 110
Thr Thr Ile Pro Asp Thr Ser Trp His Gly Ile Leu Trp Thr Thr Arg
115 120 125
Ser Asn Gln Ile Val Glu Gln Gly Leu Ile Asn Asn Val Gln Gln Ile
130 135 140
Gly Ile His Leu Ala Thr Asp Phe Tyr Leu Pro Phe Glu Leu Ile Ser
145 150 155 160
Leu Ile Leu Val Val Ser Leu Ile Gly Ile His Phe Asp Asn Gly Val
165 170 175
Lys Asp Asn Arg Ile Leu Glu Leu Thr Ile Leu Ser Lys Trp Leu Thr
180 185 190
Pro Ser Ile Asn Leu Phe Gln Glu Asn Ser Asn Ser Ser Ile Asn Ser
195 200 205
Tyr Glu Phe Leu Thr Asn Ala Ile Ser Ser Ser Ser Ser Leu Leu Pro
210 215 220
Leu His Ser Arg Thr Asn Val Arg Leu Ala Arg Gly Asn Leu Cys Thr
225 230 235 240
Pro Pro Leu Pro Phe Gly Arg Pro Thr Pro His Arg Asn Cys Leu Pro
245 250 255
Glu Thr Val Pro Trp Pro Ala Gly Leu Thr Gln Gly Thr Leu Leu Leu
260 265 270
Lys Val Val Arg Ala Phe Pro Gly Ser Met Ala Trp Val Thr Tyr Phe
275 280 285
Ser Ala Val Ala Pro Gly Met Ser Leu Val Glu Lys Gln Trp Leu Val
290 295 300
His Gly Ala His Thr Ser Ala Leu Gln Arg Leu Val Leu Gly Pro Arg
305 310 315 320
Leu Glu Ala Phe Ser Leu Pro Leu Leu Thr Leu Lys Lys Gln Gly His
325 330 335
Leu Val Ser Leu Asn Leu
340
<210> 27
<211> 21
<212> DNA
<213> maize
<400> 27
atgacgagtt ttcacgtccg a 21
<210> 28
<211> 22
<212> DNA
<213> maize
<400> 28
cgttcacgcg atttttcaag tg 22

Claims (25)

1. A method of identifying a plant with increased disease resistance, the method comprising:
a. detecting the nucleotide sequence similar to SEQ ID NO: 4-10 or 13-16, or a SNP or indel as shown in figure 1; and
b. identifying the plant as having a QTL allele, wherein the plant has increased disease resistance.
2. The method of claim 1, further comprising counter-selecting the plant from a breeding program.
3. A method of identifying a plant with increased disease resistance, the method comprising:
a. detecting in the genome of the plant any one of:
i. a polynucleotide encoding a polypeptide having the sequence of SEQ ID NO: 1-3, or a polypeptide of an amino acid sequence set forth in any one of seq id nos;
ii a polynucleotide encoding a polypeptide having an amino acid sequence substantially identical to that of SEQ ID NO: 1-3, a polypeptide having an amino acid sequence that is at least 90% identical to any one of seq id nos;
a polynucleotide comprising SEQ ID NO: 4-10; or
(iii) one or more marker alleles within 5cM of (i) or (ii) linked and associated with (i) or (ii); and
b. identifying the plant as having increased disease resistance if any of (i), (ii), or (iii) is detected.
4. A method of increasing disease resistance in a plant, the method comprising expressing in a plant a recombinant polynucleotide encoding a polypeptide having an amino acid sequence identical to SEQ ID NO: 1-3, having at least 90% sequence identity to an amino acid sequence set forth in any one of seq id nos; wherein a plant expressing a recombinant polypeptide has increased disease resistance in said plant when compared to a control plant not comprising said recombinant polynucleotide.
5. The method of claim 4, wherein the recombinant polynucleotide further comprises a heterologous promoter.
6. The method of claim 4, further comprising obtaining a progeny plant derived from a plant expressing the recombinant polynucleotide, wherein the progeny plant comprises the recombinant polynucleotide in its genome and exhibits increased disease resistance as compared to a control plant not comprising the recombinant polynucleotide.
7. The method of claim 4, wherein the plant is selected from the group consisting of: arabidopsis, maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
8. The method of claim 4, wherein the plant is a monocot.
9. A method of identifying a ZmMM1 gene variant that confers increased disease resistance to a plant, comprising the steps of:
a. combining, by gene shuffling, one or more nucleotide sequences encoding the amino acid sequence of SEQ ID NO: 1-3, and one or more fragments of any one of SEQ ID NOs: 1-3 or a fragment thereof that is at least 90% identical to any one of; and
b. identifying variants exhibiting increased disease resistance.
10. The method of claim 9, wherein the method further comprises the steps of:
a. introducing into a regenerable plant cell a recombinant construct comprising a ZmMM1 gene variant identified by the method of claim 9;
b. after step (a), regenerating a transgenic plant from the regenerable plant cell, wherein the transgenic plant comprises in its genome a recombinant DNA construct; and
c. selecting the transgenic plant of (b), wherein said transgenic plant comprises said recombinant DNA construct and exhibits increased disease resistance when compared to a control plant not comprising said recombinant DNA construct.
11. The method of claim 9 or 10, wherein the plant is selected from the group consisting of: arabidopsis, maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
12. The method of claim 9 or 10, wherein the plant is a monocot.
13. The method of claim 12, wherein said monocot is maize.
14. A method of identifying an allelic variant of the ZmMM1 gene, wherein the allelic variant is associated with increased disease resistance, the method comprising the steps of:
a. obtaining a population of plants, wherein the plants exhibit different levels of disease resistance;
b. evaluation of the expression vector for the coding sequence comprising SEQ ID NO: 1-3, or in a genomic region that regulates expression of a polynucleotide encoding the protein;
c. correlating allelic variation with increased disease resistance; and
d. identifying allelic variants associated with increased disease resistance.
15. The method of claim 14, further comprising detecting the allelic variant associated with increased disease resistance, and selecting a plant if the allelic variant is detected.
16. A method of introducing an allelic variant of the ZmMM1 gene, wherein the allelic variant is associated with increased disease resistance, the method comprising introducing a mutation in an endogenous ZmMM1 gene using a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas system, or a meganuclease, such that the allelic variant comprises a polynucleotide sequence encoding a protein at least 90% identical to any of SEQ ID NOs: 1-3.
17. A recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein said polynucleotide comprises a nucleic acid sequence encoding a polypeptide having a sequence that when operably linked to the sequence of SEQ ID NO: 1-3, which have at least 90% sequence identity when compared.
18. The recombinant DNA construct of claim 17, wherein said at least one regulatory sequence is a promoter functional in a plant cell.
19. The recombinant DNA construct of claim 17, wherein said polynucleotide comprises a sequence identical to SEQ ID NO: 4-10, having at least 95% sequence identity.
20. A transgenic plant cell comprising the recombinant DNA construct of claim 17.
21. A transgenic plant comprising the transgenic plant cell of claim 20.
22. The transgenic plant of claim 21, wherein the transgenic plant is selected from the group consisting of: arabidopsis, maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
23. A transgenic seed produced by the transgenic plant of claim 21.
24. A method of identifying and/or selecting a plant with increased disease resistance, the method comprising:
a. screening the population for a marker located on chromosome 7, the interval on chromosome 7 comprising and flanked by the primer sequences SEQ ID NO: marker M2 and the primer sequences SEQ ID NO: 11 and 12 is shown as marker M3,
b. selecting at least one plant comprising an allele of said gene from said population.
25. The method of claim 24, the method further comprising:
c. crossing the plant of (b) with a second plant; and
d. obtaining a progeny plant having the gene allele.
CN202011200759.6A 2020-01-15 2020-10-30 Method for identifying, selecting and generating disease resistant crops Pending CN113121664A (en)

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