CN1359425A - Methods - Google Patents

Abstract

The invention discloses methods which are particularly sensitive for detecting low frequencies of mutations in mitochondrially encoded genes, such as the cytochrome b gene, making these an especially useful and commercially important way of screening plant pathogenic fungi for the onset of fungicidal resistance wherein the resistance is due to a mutation in a mitochondrially encoded gene. The methods disclosed include single nucleotide polymorphism detection techniques especially PCR detection methods. Also disclosed are DNA sequences encoding part of the wild type and mutant cytochrome b sequences of a range of plant pathogenic fungi and the use of the sequence information to detect mutations giving rise to fungicidal resistance. Allele specific oligonucleotides and oligonucleotide probes, diagnostic primers and diagnostic kits are also disclosed.

Description

Method

Invention field

The present invention relates to any (or a kind of) mononucleotide polymorphic detection technique, preferably use amplification to resist abruptly-changing system (ARMS) and detect the diagnostic method that causes in the fungi the cytochrome b sudden change of the compound resistance in strobilurins analogue or the same crossed resistance group.The present invention also relates to be used for the sudden change Auele Specific Primer of described method and the diagnostic kit that contains these primers.The invention still further relates to and cause containing of the evaluation of the fungi of specific sudden change in fungal cell's pigment b gene the described sudden change of the compound resistance in strobilurins analogue or the same crossed resistance group.

Background of invention

The widespread use of mycocide is a kind of phenomenon relatively recently in agricultural, and most of main development occur in the period of nearest 40.Before, the peasant ignored usually or did not recognize the influence of fungal pathogens to its crop yield and quality.Yet these losses now are unacceptable, and the peasant relies on the application Fungicidal compounds to prevent and treat fungal disease.Therefore, commercial mycocide has become a commercial important component part of whole farmingizations, the sales volume Yue Wei $59 hundred million in the whole world in 1996, equal 18.9% (the Wood Mackenzie in whole farmingization market, 1997a ' Agchem products-The key agrochemical product groups ', Agrochemical Service, Update of the Products Section, in May, 1997,1-74).The peasant can obtain many mycocides; Latest edition The Pesticide Manual (Tomlin, 1994, the 10 editions, British Crop Protection Council, Farnham, UK and the Royal Society of Chemistry, Cambridge UK) comprises 158 kinds of different fungicidal effective constituents of present use.Yet, target for find and the further industrial research of exploitation new compound extremely extensive, and the artefact management program is ensureing that best, the most permanent production performance avoids of crucial importance aspect the infringement of the mycocide that has the specific function mode and/or belong to a specific compounds.Specifically, it is essential and when introducing mycocide, develop effective resistance operating strategy (resistance managementstrategies) (Fungicide Resistance Management:Into The Next Millenium (Russell) 1999 with new mode of action, be stated from Pesticide Outlook, in October, 1999 (213-215).

The strobilurins analogue has constituted the novel main agricultural fungicides of a class, and they since the 4-triazole, have been considered to the most exciting development in agricultural fungicides aspect since finding 1,2 in nineteen seventies.

The Fungicidally active of strobilurins analogue is its result who suppresses the ability of mitochondrial respiratory in the fungi.More particularly, determine, these compounds have new single site binding mode, by the blocking-up reduced coenzyme Q: cytochrome c oxido-reductase composite (cytochrome b c1) and fungi is brought into play its influence, reduce the generation (Becker etc., FEBS Letts.132 329-33) of high energy ATP among the fungal cell thus.This class inhibitor stops ubiquinone redox site Q on the polymer cytochrome b albumen ₀Electron transport (1993Biochim.et Biophys.Acta 243-271 such as Esposti).Different with many mitochondrial proteins, cytochrome b albumen is the plastosome coding.

Report in the document shows that the specific amino acids on the cytochrome b target site changes the activity that can influence the strobilurins analogue.At yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) (1989 J.Biol.Chem.264 such as JP Rago, 14543-14548), mouse (1988 J.Mol.Biol.203 such as Howell, 607-618), Lay is breathed out clothing trichomonad (Chlamydomonasreinhardtii) (1991 Genetics 127 such as Bennoun, 335-343) and plant in the uncertain red bacterium (Rhodobacterspp) (1989 EMBO such as Daldal J.3951-3961) of name, carried out deep mutagenesis research.Also from intend positive sea urchin (Paracentrotus lividus) (Esposti etc. in Echinoidea, 1990 FEBS 263,245-247) and Basidiomycetes (Basidiomycete) the fungi breast little mushroom of handle (Mycena galopoda) and the Strobilurus tenacellus (Kraiczy etc. that produce strobilurins analogue natural variation body, 1996, Eur.J.Biochem.235, research has been collected relevant information to the natural foundation of strobilurins analogue resistance 54-63).Two specific regions are arranged in the cytochrome b gene, and amino acid whose change has remarkably influenced to strobilurins analogue activity in these two zones.These zones comprise amino-acid residue 125-148 and 250-295 (according to the residue numbering system of yeast saccharomyces cerevisiae).Show, more accurate residue 126,129,132,133,137,142,143,147,148,256,275 and 295 amino acid change produce resistance (1996 Biochim.Biophys.Acta 1275 such as Brasseur to the strobilurins analogue, 61-69 and Esposti etc. (1993) Biochimica et Biophysica Acta, 1143,243-271).

The present invention has identified the critical importance of one of these sudden changes in the cytochrome b gene that shows the important phytopathogenic fungi field isolate that the compound in strobilurins analogue or the same crossed resistance group is had resistance for the first time.

Brief summary of the invention

According to first aspect of the present invention, we provide a kind of method that fungal nucleic acid suddenlys change that is used for detecting, the existence of wherein said sudden change causes the resistance of fungi to any other compound in strobilurins analogue or the same crossed resistance group, and described method comprises with any (or a kind of) mononucleotide polymorphic detection technique identifying whether there is described sudden change in fungal nucleic acid.

In the present invention, we have designed the novel diagnostic method that is used for detecting based on mononucleotide polymorphic detection method (comprising allele specific amplification) point mutation of fungal cell's pigment b gene now.It will be apparent to one skilled in the art that many analytical procedures can be used for detecting the Nucleotide according to whether there being variation on one or more polymorphic positions of the present invention.Generally speaking, need suddenly change authentication technique, optional amplified reaction and optional signal generation system of the detection of allelic variation.Nollau etc., Clin.Chem.43,1114-1120,1997 and standard textbook Laboratory Protocols for Mutation Detection for example, buU.Landegren writes, Oxford University Press, 1996 and the PCR of Mewton and Graham the 2nd edition, BIOS Scientific Publishers limited has summarized the many methods that are used to detect allelic variation at present in 1997.The allele specific amplification reaction comprises the method based on primer, the method that comprises PCR-based, more particularly, comprise allele-specific polymerase chain reaction (PCR) extension (ASPCR), specifically be ARMS (the mutagenesis system is resisted in amplification), wherein said sudden change produces the resistance to the strobilurins analogue, and these are particularly preferred in the method for the present invention.Method of the present invention also comprises indifference (indiscriminate) PCR, then the amplicon that is produced is carried out specificity and surveys.These methods are suitable for detecting the specific alleles that can give any other compound resistance in any strobilurins analogue or the same crossed resistance group.Developed the reinforcement test (robust test) that is used for detecting this point mutation at multiple fungal plant pathogen.When the resistance of also giving for a kind of resistance mechanism of compound another kind of compound, even when the described mode of action is inequality, can think that also compound belongs to same crossed resistance group.The technical description of ASPCR is in U.S. Patent number 5639611, and the ARMS versatility is described among the european patent number EP 332435.

Other single polymorphic detection technique that can be used for detecting sudden change comprises that for example restriction fragment length polymorphism (RFLP), single strand conformation polymorphism, polyclone analysis, allele specific oligonucleotide hybridization, mononucleotide primer extend (Juvonen etc., (1994) Hum Genet 9316-20; Huoponen etc., (1994) Hum Mutat 329-36; Mashima etc. (1995), Invest Opthelmol.Vision.Sci 36,1714-20; Howell etc. (1994) Am J HumGenet.55 203-206; Koyabashi etc., (1994) Am.J.Hum Genet.55 206-209; Johns and Neufeld (1993) Am J Hum Genet 53 916-920; Chomyn etc., (1992) Proc.Natl.Acad.Sci USA 89 4221-4225) and Invader ^TMTechnology (can derive from ThirdWave Technologies Inc.502 South Rosa Road, Madison, WI 53719USA).

Preferably use the detection system of PCR-based.

A preferred embodiment according to first aspect present invention, we are provided for detecting the method for suddenling change in the fungal nucleic acid, the existence of wherein said sudden change causes the resistance of fungi to any other compound in strobilurins analogue or the same crossed resistance group, described method comprises the existence of the amplicon that detection produces between the reaction period at PCR, wherein said PCR reaction is included in suitable Nucleotide triphosphoric acid and is used for polymeric reagent and exists down, the given the test agent that comprises fungal nucleic acid is contacted with a kind of primer, whether exist described sudden change directly related in the detection of wherein said amplicon and the described nucleic acid.

The detection of the amplicon that produces between the reaction period at PCR can directly depend on the extension that has specific primer for described sudden change, promptly wherein primer extension depends on the existence of described sudden change, therefore only described primer combination and/or when being extended when described sudden change exists, just produce amplicon (also is like this for the ARMS technical situation), equally, it can directly depend on the extension that does not have (for example wild-type sequence) specific primer for described sudden change, maybe can it is directly related with the PCR extension products that contains described mutant DNA sequence (promptly wherein detecting the amplicon that comprises described mutant DNA sequence).Preferred especially first kind of alternative method.

Described amplicon can derive from arbitrary PCR circulation, and this comprises the first allele-specific primers extension products.

In another preferred embodiment, the invention provides a kind of method that fungal nucleic acid suddenlys change that is used for detecting, the existence of wherein said sudden change causes the resistance of fungi to any other compound in strobilurins analogue or the same crossed resistance group, described method is included in suitable Nucleotide triphosphoric acid and is used for polymeric reagent and exists down, the given the test agent that comprises fungal nucleic acid is contacted with a kind of suitable diagnostic primer, make and perhaps when having wild-type sequence, extended described diagnostic primer or extended when in described sample, having described sudden change; And whether exist according to the diagnostic primer extension product and to detect described sudden change and whether exist.

In a preferred embodiment again, the invention provides a kind of method that is used to detect the fungal nucleic acid sudden change, the existence of wherein said sudden change causes the resistance of fungi to any other compound in strobilurins analogue or the same crossed resistance group, described method is included in suitable Nucleotide triphosphoric acid and is used for polymeric reagent and exists down the given the test agent that comprises fungal nucleic acid is contacted with a kind of diagnostic primer of specific sudden change, makes described diagnostic primer be extended when having described sudden change in described sample; And whether exist according to the diagnostic primer extension product and to detect described sudden change and whether exist.

A particularly preferred embodiment according to first aspect present invention, we provide a kind of method that fungal nucleic acid suddenlys change that is used for detecting, the existence of wherein said sudden change causes the resistance of fungi to any other compound in strobilurins analogue or the same crossed resistance group, described method is included in suitable Nucleotide triphosphoric acid and is used for polymeric reagent and exists down the given the test agent that comprises fungal nucleic acid is contacted with a kind of diagnostic primer of specific sudden change, makes described diagnostic primer be extended when only having described sudden change in described sample; And whether exist according to the diagnostic primer extension product and to detect described sudden change and whether exist.

Term diagnostic primer used herein is used to refer to the primer that is used for identifying specifically whether sudden change or wild-type sequence exist, and the term general primer is used to refer to and is incorporated into the chain opposite with described diagnostic primer and is positioned at 3 ' DNA of described diagnostic primer institute cog region and by allow the primer of DNA amplification intervening sequence section with described diagnostic primer effect during PCR.When described diagnostic primer was the ARMS primer, it can have 3 ' mispairing when it was compared with mutant nucleotide sequence or wild-type sequence.

Of the present invention aspect this and in all others and the embodiment, preferably use as or diagnostic primer or opposite strand on the detection system of an integrated part of general primer, detect the extension of described primer extension product.This has more fully in this article describes.

Method of the present invention is particularly suitable for detecting the sudden change in the plastosome encoding gene of mycocide target protein, more specifically be suitable for detecting the sudden change in fungal cell's pigment b gene, wherein said sudden change causes suppressing for the proteic mycocide activity of cytochrome b, but still the generation of ATP allow to take place, most preferably wherein the described sudden change in fungal cell's pigment b gene causes one of following aminoacid replacement: A ₁₂₆T, F ₁₂₉L, Y ₁₃₂C, C ₁₃₃Y, G ₁₃₇R/S/E/V, W ₁₄₂T/K, G ₁₄₃A, I ₁₄₇F, T ₁₄₈M, N ₂₅₆Y/K/I, L ₂₇₅F/S/T or L ₂₉₅F, wherein in described sequence by first amino acid on the position of described numeric representation by second aminoacid replacement, the existence of described replacement causes the resistance of fungi for any other compound in strobilurins analogue or the same crossed resistance group, and the discriminating of wherein said residue is based on the residue numbering system of brewing yeast cell pigment b.

Compound in strobilurins and the same crossed resistance group comprises for example azoxystrobin, picoxystrobin, kresoxim-methyl, trifloxystrobin, famoxadone and fenamidone.

We have found that, in fungal cell's pigment b nucleic acid with brewing yeast cell pigment b sequence in the corresponding position of the 143rd codon/amino acid fungi in the isolate of strobilurins analogue resistance plant pathomycete field be key determiner for the resistance of any other compound in strobilurins analogue or the same crossed resistance group.Method of the present invention described herein be particularly suitable for detecting with brewing yeast cell pigment b residue 143 corresponding positions on sudden change, wherein glycine residue is by another kind of aminoacid replacement, this has suppressed the activity of any other compound in strobilurins analogue or the same crossed resistance group, and is having described mutant cell pigment b gene and causing thus causing resistant phenotype in the fungi of fungi to the resistance of any other compound in strobilurins analogue or the same crossed resistance group.

Described method is preferably used in and detects the sudden change that causes being selected from the above glycine residue of brewing yeast cell pigment b residue 143 corresponding positions following a kind of aminoacid replacement: arginine, Serine, halfcystine, Xie Ansuan, aspartic acid, L-glutamic acid, tryptophane most preferably are L-Ala.

In a preferred embodiment again of first aspect present invention, we provide a kind of method that is used to detect fungal cell's pigment b transgenation now, described sudden change cause in coded protein with brewing yeast cell pigment b residue 143 corresponding positions on glycine replaced (G by L-Ala ₁₄₃A), cause the resistance of fungi to any other compound in strobilurins analogue or the same crossed resistance group thus, described method comprises with any (or a kind of) mononucleotide polymorphic detection technique identifying to suddenly change described in the fungal nucleic acid whether exist.

Cause G in coded protein in fungal cell's pigment b gene ₁₄₃The sudden change that A replaces is normally gone up guanine base in second (base) of described codon and is changed into the cytosine(Cyt) base, and in this single nucleotide polymorphism of best detection aspect all and in the embodiment of the present invention described herein.

In the another preferred embodiment of first aspect present invention, we provide a kind of now and are used for detecting fungal cell's pigment b gene and cause G in the coded protein ₁₄₃The diagnostic method of the sudden change that A replaces, described method comprises the existence of the amplicon that detection PCR produced between the reaction period, whether wherein said PCR reaction is included in suitable Nucleotide triphosphoric acid and exists down the given the test agent that comprises fungal nucleic acid is contacted with the diagnostic primer with a kind of polymeric reagent that is used for, exist described sudden change directly related in the detection of wherein said amplicon and the described nucleic acid.

In a particularly preferred embodiment of first aspect present invention, we provide a kind of now and are used for detecting fungal cell's pigment b gene and cause G in the coded protein ₁₄₃The diagnostic method of the sudden change that A replaces, described method be included in suitable Nucleotide triphosphoric acid and a kind of be used for polymeric reagent exist make down the given the test agent that comprises fungal nucleic acid with for causing coded protein G ₁₄₃The diagnostic primer contact of the sudden change that A replaces makes described diagnostic primer exist in described sample and causes G in the coded protein ₁₄₃Extended during sudden change that A replaces; And whether exist according to the diagnostic primer extension product and to detect described sudden change and whether exist.

In a particularly preferred embodiment again of first aspect present invention, we provide a kind of now and are used for detecting fungal cell's pigment b gene and cause G in the coded protein ₁₄₃The diagnostic method of the sudden change that A replaces, described method be included in suitable Nucleotide triphosphoric acid and a kind of be used for polymeric reagent exist make down the given the test agent that comprises fungal nucleic acid with for causing coded protein G ₁₄₃The diagnostic primer contact of the sudden change that A replaces makes described diagnostic primer only exist in described sample and causes G in the coded protein ₁₄₃Extended during sudden change that A replaces; And whether exist according to the diagnostic primer extension product and to detect described sudden change and whether exist.

Term G used herein ₁₄₃A is used to refer to that glycine residue is replaced by alanine residue on the 143rd codon/amino acid whose position that is equal to brewing yeast cell pigment b sequence in fungal cell's pigment b sequence.Used other residue that this nomenclature is used for this paper citation changes, and promptly all quote from respect to brewing yeast cell pigment b protein sequence all positions.Brewing yeast cell pigment b gene and protein sequence can obtain (referring to EMBL accession number X84042 and SWISSPROT accession number P00163) on EMBL and SWISSPROT database.The technician will appreciate that, derive from being equal to of different plant species proteic precise length and record may since N-terminal or C-terminal and/or one or more inner disappearance or insertion change.Owing to contain corresponding to G in the yeast saccharomyces cerevisiae ₁₄₃The aminoacid sequence section of residue be very conservative (Proc.Nat.Acad.Sci. such as Widger, U.S.A.81 (1984) 674-678), therefore simply be to detect or use one of several sequence alignment programs (comprising Megalign or Macaw) by range estimation to identify residue accurately corresponding in the new fungal cell's pigment b sequence that obtains.Though specify G in this application ₁₄₃, but because position equivalents and functional equivalent body are arranged, the exact position of this glycine may not be the 143rd residue from its N-terminal in new cytochrome b.The consensus sequence of brewing yeast cell pigment b is provided in SWISSPROT accession number P00163.Described herein of the present invention aspect all and in the embodiment, the position in the cytochrome b sequence is preferably determined with respect to the brewing yeast cell pigment b sequence that provides among the EMBL accession number X84042.Perhaps, described herein of the present invention aspect all and in the embodiment, the position in the cytochrome b sequence is preferably determined with respect to the consensus sequence of the brewing yeast cell pigment b that provides among the SWISSPROT accession number P00163.

According to one aspect of the present invention, be provided for diagnosing the method for single nucleotide polymorphism in fungal cell's pigment b gene, described method comprises mensuration with respect to the fungal nucleic acid sequence on the correspondence position of the one or more bases in the locational amino acid whose triplet corresponding with brewing yeast cell pigment b residue 143 in the described cytochrome b albumen of coding, and determines the resistance situation of described fungi to the compound in strobilurins analogue or the same crossed resistance group according to the polymorphism in the described cytochrome b gene.

Described herein of the present invention aspect all and in the embodiment, preferably the coding described cytochrome b albumen in brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in only a base show sudden change, promptly only on a position, there is single nucleotide polymorphism, further preferably on first base of described triplet or second base, have single nucleotide polymorphism on second base of the most described triplet.

A preferred embodiment according to this aspect of the present invention, be provided for diagnosing the method for single nucleotide polymorphism in fungal cell's pigment b gene, described method comprises the fungal nucleic acid sequence that is determined in the described cytochrome b albumen of coding corresponding on the correspondence position of second base of the locational amino acid whose triplet of brewing yeast cell pigment b residue 143, and determines the resistance situation of described fungi to the compound in strobilurins analogue or the same crossed resistance group according to the polymorphism in the described cytochrome b gene.

In the embodiment aspect the present invention is above-mentioned, described diagnostic method described herein is to be to have G and/or C corresponding to the single nucleotide polymorphism on the correspondence position of second base in the locational amino acid whose triplet of brewing yeast cell pigment b residue 143 in the described cytochrome b albumen of coding in described DNA wherein.

First	Second		The 3rd
				5 ' end	C	G	3 ' end
G	L-Ala	Glycine	U
				L-Ala	Glycine	C
	L-Ala	Glycine	A
				L-Ala	Glycine	G

Table 1: codon is selected

Glycine to the point mutation of L-Ala requires that G becomes C on second base of described codon.Other sudden change also can produce on the 3rd of described codon, and this is because the degeneracy (referring to table 1) of L-Ala and glycine genetic code, but this considers when the described diagnostic primer of design easily.The diagnostic primer is the ARMS primer preferably.(Newton etc. have described the notion of ARMS primer comprehensively among NucleicAcid Research 17 (7) 2503-2516 1989).Therefore, can design the ARMS primer, be used on wild-type strobilurins analogue responsive type cytochrome b gene, measuring G ₁₄₃Unique sequence information that the A point mutation produces.Do not need to obtain because G ₁₄₃The resistance isolate in new purpose fungi that the A sudden change produces.Some example of corresponding plants pathomycete is listed in the table 2.This table does not also mean that it is detailed.The phytopathologist can easily identify those fungies relevant with the inventive method.

	Can analyze G 143The example of the bacterial classification of A:
		1	Grape is given birth to single shaft mould (Plasmopara viticola)
2	Standing grain powdery mildew wheat/barley mutation (Erysiphe graminis f.sp.tritici/hordei that causes a disease
		3	Rye beak spore (Rhynchosporium secalis)
4	Circle nuclear cavity bacteria (Pyrenophora teres)
		5	Standing grain green-ball chamber bacterium (Mycosphaerella graminicola)
6	Mycosphaerella?fijiensis?var.difformis

??7	Monofilament shell (Sphaerotheca fuliginea)
		??8	Grape snag shell (Uncinula necator)
??9	Standing grain is given birth to thorn dish spore (Colletotrichum graminicola)
		??10	Melon and fruit corruption mould (Pythium aphanidermatum)
??11	Colletotrichum gloeosporiodes (Colletotrichum gloeosporioides)
		??12	Tomato powder spore (Oidium lycopersicum)
??13	Tartar's internal thread powdery mildew (Leveillula taurica)
		??14	The false downy mildew (Pseudoperonospora cubensis) of Cuba
??15	Upright withered chain lattice spore (Alternaria solani)
		??16	Semen arachidis hypogaeae tail spore (Cercospora arachidola)
??17	Dry thread Pyrenomycetes (Rhizoctonia solani)
		??18	Venturia inaequalis (Venturia inaequalis)
??19	??Magnaporthe?grisea
		??20	Phytophthora infestans (Phytophora infestans)
??21	??Mycosphaerella?musicola

Table 2: wherein can analyze G ₁₄₃The bacterial classification example of A

The inventive method described herein is particularly useful for phytopathogenic fungi, and in particular for following fungi strain: grape is given birth to single shaft mould (Plasmopara viticola), the mutation (Erysiphe graminis f.sp.tritici/hordei) of causing a disease of standing grain powdery mildew wheat/barley, rye beak spore (Rhynchosporium secalis), circle nuclear cavity bacteria (Pyrenophora teres), standing grain green-ball chamber bacterium (Mycosphaerella graminicola), venturia inaequalis (Venturia inaequalis), Mycosphaerella fijiensis var.difformis, monofilament shell (Sphaerothecafuliginea), grape snag shell (Uncinula necator), standing grain is given birth to thorn dish spore (Colletotrichumgraminicola), melon and fruit corruption mould (Pythium aphanidermatum), Colletotrichum gloeosporiodes (Colletotrichum gloeosporioides), tomato powder spore (Oidium lycopersicum), Magnaporthe grisea, phytophthora infestans (Phytophthora infestans), Tartar's internal thread powdery mildew (Leveillula taurica), the false downy mildew (Pseudoperonospora cubensis) of Cuba, upright withered chain lattice spore (Alternaria solani), dry thread Pyrenomycetes (Rhizoctonia solani), Mycosphaerella musicola and Semen arachidis hypogaeae tail spore (Cercospora arachidola).

More on the one hand, the invention provides and be used for detecting the method for fungi the resistance of strobilurins analogue or any other compound of same crossed resistance group, described method comprises identifying in the fungal nucleic acid whether have sudden change, the existence of wherein said sudden change causes the resistance to any other compound in strobilurins analogue or the same crossed resistance group, described method comprise evaluation in the described cytochrome b albumen of coding with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in whether have single nucleotide polymorphism on the correspondence position of one or more bases.

In the preferred embodiment again aspect this, the invention provides and be used for detecting the resistance of fungi for strobilurins analogue or any other compound of same crossed resistance group, described method comprises identifies whether fungal nucleic acid exists sudden change, the existence of wherein said sudden change causes the resistance to any other compound in strobilurins analogue or the same crossed resistance group, described method comprise evaluation in the described cytochrome b albumen of coding with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in whether have single nucleotide polymorphism on the correspondence position of second bit base.

In the preferred embodiment of the present invention aspect this, adopt any (or a kind of) mononucleotide polymorphic detection technique, identify in the cytochrome b gene of fungal nucleic acid whether have single nucleotide polymorphism on the correspondence position of second bit base in the amino acid whose triplet on the coding and brewing yeast cell pigment b residue 143 corresponding positions.

The present invention also provides all or part of wild-type cell pigment b of coding proteic fungal DNA sequence, wherein said dna sequence dna in described wild-type protein with the corresponding position of brewing yeast cell pigment b residue 143 on the glycine residue of encoding, wherein said sequence can derive from or derive from selects following a kind of fungi: it is mould that grape is given birth to single shaft, the mutation of causing a disease of standing grain powdery mildew wheat/barley, rye beak spore, the circle nuclear cavity bacteria, standing grain green-ball chamber bacterium, Mycosphaerella fijiensis var.difformis, the monofilament shell, grape snag shell, standing grain is given birth to thorn dish spore, the melon and fruit corruption is mould, Colletotrichum gloeosporiodes, the tomato powder spore, Tartar's internal thread powdery mildew, the false downy mildew of Cuba, upright withered chain lattice spore, dry thread Pyrenomycetes, Mycosphaerella musicola and Semen arachidis hypogaeae tail spore.

Be preferably among the described DNA any one or both sides according to the fungal DNA sequence of the above-mentioned aspect of the present invention corresponding to the position of one or more bases in the described triplet, be preferably in the encoding said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in any one or both sides of second bit base, contain 30 Nucleotide of having an appointment, because nucleic acid that should the zone provides design species specificity and sudden change specific reagent for the technician and/or has been used for the necessary all information of method of all mononucleotide polymorphic detection techniques.About 30 of term used herein is meant that described sequence can comprise 30 Nucleotide at the most, and for example 5 to 10,15,20 or 25 Nucleotide maybe can comprise the Nucleotide more than 30.

Be used to refer to dna sequence dna or protein sequence or their fragment at term used herein " all or part of " aspect all dna sequences and the protein sequence.DNA or proteic fragment can for example be 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% of described full length sequences.

To those skilled in the art, obviously containing the sample of genome (plastosome) DNA and cDNA all can be according to analysis of the present invention.When needs are considered to contain the sample of genomic dna intron group structure, use described sequence information.The example that comprises the wild-type fungal DNA sequence of part wild-type cell pigment b gene order according to the above-mentioned aspect of the present invention provides in following table, and described sequence constitutes one side more of the present invention.

Species	Sequence
		Grape is given birth to single shaft mould (cDNA and genomic dna)	5’TTTTGCCTTGGGGACAAATGAGTTTTTGGG GTGCAAC AGTTATTACAAATTTATTCTCGGC3’(SEQ?ID?NO?1)
The mutation (cDNA and genomic dna) of causing a disease of standing grain powdery mildew wheat/barley	5’TATTGCCATACGGGCAGATGAGCCACTGGG GTGCAAC CGTTATCACTAACCTAATGAGCGC3’(SEQ?ID?NO?2)
		Rye beak spore (cDNA and genomic dna)	5’TGCTTCCTTATGGACAGATGTCTTTATGAG GTGCCAC AGTTATAACTAATCTTATGAGTGC?3’(SEQ?ID?NO?3)
Circle nuclear cavity bacteria (cDNA)	5’TTTTACCCTACGGGCAAATGAGCCTTTGAG GTGCTAC AGTTATTACTAACCTTATGAGTGC3’(SEQ?ID?NO?4)
		Circle nuclear cavity bacteria (genomic dna)	5’TTTTACCCTACGGGCAAATGAGCCTTTGAG GTGAAAT ATTTGCCTCAAATGTATAACTAAT3’(SEQ?ID?NO?5)
Standing grain green-ball chamber bacterium (cDNA and genomic dna)	5’TATTACCTTATGGTCAAATGTCTTTATGAG GAGCAAC AGTTATAACTAACTTATTGAGTGC3’(SEQ?ID?NO?6)
		Mycosphaerella fijiensis var.difformis (cDNA and genomic dna)	5’TTTTACCTTATGGTCAAATGTCTTTATGA GGAGCTACA GTTATAACTAATTTAATGAGCGC3’(SEQ?ID?NO?7)
Monofilament shell (cDNA)	5’TACTTCCCTTCGGTCAAATGTCGCTCTGGG GTGCAAC CGTTATTACTAACCTTATGAGCGC3’(SEQ?ID?NO?8)
		The monofilament shell (genomic dna- *Available upstream 6bp)	5’ *TCTGGG GTGCAACCGTTAAGTAATAGCGGTTGTAAAA (SEQ?ID?NO?9)
Grape snag shell (cDNA)	5’TTTTACCCTACGGGCAGATGAGCCTATGGG GTGCAAC CGTTATTACTAACCTTATGAGCGC3’(SEQ?ID?NO?10)
		Grape snag shell (genomic dna- *Available upstream 10bp)	5’ *AGCCTATGGG GTGCAACCGTTAAGTAGGTAATAGCG GTTGA3’(SEQ?ID?NO?11)
Standing grain is given birth to thorn dish spore (genomic dna and cDNA)	5’TTTTACCTTACGGACAAATGTCATTATGAG GTGCTAC AGTTATTACTAACCTTATVGTGC3’(SEQ?ID?NO?12)
		Melon and fruit corruption mould (genomic dna and cDNA)	5’TATTACCTTGGGGTCAAATGAGTTTTTGGG GTGCTAC TGTTATTACTAATTTATTTTCAGC3’(SEQ?ID?NO?13)
Colletotrichum gloeosporiodes (genomic dna and cDNA)	5’TTTTACCTTATGGACAAATGTCATTATGAG GTGCAAC AGTTATTACTAACCTTATAAGTGC3’(SEQ?ID?NO?14)
		Tomato powder spore (cDNA)	5’TTTTACCCTACGGGCAGATGAGCCTGTGGG GTGCAAC CGTTATTACTACCTTATGAGCGC3’(SEQ?ID?NO?15)
Tartar's internal thread powdery mildew	5’TTTTACCATACGGACAAATGTCATTATGAG GTGCAAC

(cDNA)	AGTTATTACTAACCTTATGAGTGC3’(SEQ?ID?NO?16)
		The false downy mildew (cDNA and genomic dna) of Cuba	5’TTTTACCTTGGCGACAAATGAGTTTTTGGGGTGCAAC TGTTATTACTAATTTATTTTCTGC3’(SEQ?ID?NO?17)
Upright withered chain lattice spore (cDNA and genomic dna)	5’TTCTTCCTATGGGCAAATGTCTTTATGAGGTGCTACA GTTATTACTAACCTTATGAGTGC3’(SEQ?ID?NO?18)
		Semen arachidis hypogaeae tail spore (cDNA and genomic dna)	5’TATTACCTTATGGACAAATGTCATTATGAGGAGCTAC AGTTATTACTAATTTATTATCTGC3’(SEQ?ID?NO?19)
Dry thread Pyrenomycetes (cDNA)	5’TGCTTCCATACGGGCAAATGTCTCTGTGGGGTGCTAC AGTAATTACTAATTTACTTTCTGC3’(SEQ?ID?NO?20)
		Mycosphaerella musicola (genomic dna and cDNA)	5’TTTTACCTTATGGTCAAATGTCTTTATGAGGAGCTACA GTTATAACTAATTTAATGAGTGC3’(SEQ?ID?NO?21)

Table 3

In last table, cause normal glycine residue to be shown that with runic and following underscore the resistance to the compound in strobilurins analogue or the same crossed resistance group is given in wherein said replacement in the amino acid whose triplet on coding and brewing yeast cell pigment b residue 143 corresponding positions by second bit base of an alternative aminoacid replacement.

The present invention also extends to and shows the fungal DNA sequence that homology or sequence identity are arranged with dna sequence dna described in the table 3, for example comprises the variation of the dna sequence dna of finding in the different samples of same species or isolate.These variations can be for example owing to using alternative codon selection, change intron/exon plastosome group structure and aminoacid replacement to cause.

More on the one hand, the invention provides the proteic fungal DNA sequence of all or part of cytochrome b of coding, when the proteic wild-type dna sequence dna of described sequence aligning Codocyte pigment b is arranged, can observe described sequence in described DNA in the code for said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet on the correspondence position of one or more bases, contain a polymorphic sudden change of Nucleotide, described sudden change causes normal glycine residue by an alternative aminoacid replacement, and prerequisite is that described dna sequence dna is not the little mushroom sequence of newborn handle of Codocyte pigment b.

In the preferred embodiment again aspect this, the invention provides the proteic fungal DNA sequence of all or part of cytochrome b of coding, when the proteic wild-type dna sequence dna of described sequence aligning Codocyte pigment b is arranged, can observe described sequence in described DNA in the code for said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet on the correspondence position of second bit base, contain a polymorphic sudden change of Nucleotide, described sudden change causes normal glycine residue by an alternative aminoacid replacement, and prerequisite is that described dna sequence dna is not the little mushroom sequence of newborn handle of Codocyte pigment b.

Fungal DNA sequence according to the above-mentioned aspect of the present invention is preferably among the described DNA for any one or both sides of answering the position of one or more bases in the described triplet, be preferably in the encoding said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in any one or both sides of second bit base, contain 30 Nucleotide of having an appointment, because nucleic acid that should the zone provides design species specificity and sudden change specific reagent for the technician and/or has been used for the necessary all information of method of all mononucleotide polymorphic detection techniques.About 30 of term used herein is meant that described sequence can comprise 30 Nucleotide at the most, and for example 5 to 10,15,20 or 25 Nucleotide maybe can comprise the Nucleotide more than 30.

The present invention also provides all or part of mutant cell pigment b of coding proteic fungal DNA sequence, the resistance for the compound in strobilurins analogue or the same crossed resistance group is given in the existence that suddenlys change among the wherein said DNA, described sudden change betide among the described DNA in the code for said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet on the correspondence position of one or more bases, prerequisite is that described dna sequence dna is not the little mushroom sequence of newborn handle of Codocyte pigment b.

In a preferred embodiment aspect this, the present invention also provides all or part of mutant cell pigment b of coding proteic fungal DNA sequence, the resistance to the compound in strobilurins analogue or the same crossed resistance group is given in the existence that suddenlys change among the wherein said DNA, described sudden change betide among the described DNA in the code for said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in the correspondence position of second bit base on, prerequisite is that described dna sequence dna is not the little mushroom sequence of newborn handle of Codocyte pigment b.

Of the present invention above-mentioned aspect in, occur among the described DNA in the code for said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in the correspondence position of second bit base on sudden change, preferably guanine base becomes the cytosine(Cyt) base.

Preferably can derive from or derive from according to the proteic fungal DNA sequence of all or part of mutant cell pigment of the coding of the above-mentioned aspect of the present invention b and be selected from following a kind of fungi: it is mould that grape is given birth to single shaft, the mutation of causing a disease of standing grain powdery mildew wheat/barley, rye beak spore, the circle nuclear cavity bacteria, standing grain green-ball chamber bacterium, venturia inaequalis, Mycosphaerella fijiensis var.difformis, the monofilament shell, grape snag shell, standing grain is given birth to thorn dish spore, the melon and fruit corruption is mould, Colletotrichum gloeosporiodes, the tomato powder spore, Magnaporthe grisea, phytophthora infestans, Tartar's internal thread powdery mildew, the false downy mildew of Cuba, upright withered chain lattice spore, dry thread Pyrenomycetes, Mycosphaerella musicola and Semen arachidis hypogaeae tail spore, the existence that suddenlys change among the wherein said DNA are given the resistance for the compound in strobilurins analogue or the same crossed resistance group.

The present invention also extends to the dna sequence dna that the sequence that provides in all or part of table 3 is provided, wherein in described DNA in the code for said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in the correspondence position of second bit base on residue be cytosine(Cyt) residue (SEQ ID NO 176-196).These sequences are shown in following, and constitute one side: 5 ' TTTTGCCTTGGGGACAAATGAGTTTTTGGGCTGCAACAGTTATTACAAATTTATTC TCGGC3 ' (SEQ ID NO 176) more of the present invention.

5’TATTGCCATACGGGCAGATGAGCCACTGGGCTGCAACCGTTATCACTAACCTAA

TGAGCGC3’-?(SEQ?ID?NO?177)

5’TGCTTCCTTATGGACAGATGTCTTTATGAGCTGCCACAGTTATAACTAATCTTAT

GAGTGC3’-(SEQ?ID?NO?178)

5’TTTTACCCTACGGGCAAATGAGCCTTTGAGCTGCTACAGTTATTACTAACCTTAT

GAGTGC3’-(SEQ?ID?NO?179)

5’TTTTACCCTACGGGCAAATGAGCCTTTGAGCTGAAATATTTGCCTCAAATGTATA

ACTAAT3’-(SEQ?ID?NO?180)

5’TATTACCTTATGGTCAAATGTCTTTATGAGCAGCAACAGTTATAACTAACTTATTGAGTG

C3’(SEQ?ID?NO?181)

5’TTTTACCTTATGGTCAAATGTCTTTATGAGCAGCTACAGTTATAACTAATTTAAT

GAGCGC3’-(SEQ?ID?NO?182)

5’TACTTCCCTTCGGTCAAATGTCGCTCTGGGCTGCAACCGTTATTACTAACCTTAT

GAGCGC3’-(SEQ?ID?NO?183)

5’ ^*TCTGGGCTGCAACCGTTAAGTAATAGCGGTTGTAAAA-(SEQ?ID?NO?184)

5’TTTTACCCTACGGGCAGATGAGCCTATGGGCTGCAACCGTTATTACTAACCTTAT

GAGCGC3’-(SEQ?ID?NO?185)5’ ^*AGCCTATGGGCTGCAACCGTTAAGTAGGTAATAGCGGTTGA3’-(SEQ?ID?NO?186)5’TTTTACCTTACGGACAAATGTCATTATGAGCTGCTACAGTTATTACTAACCTTATAAGTGC3’-(SEQ?ID?NO?187)5’TATTACCTTGGGGTCAAATGAGTTTTTGGGCTGCTACTGTTATTACTAATTTATTTTCAGC3’-(SEQ?ID?NO?188)5’TTTTACCTTATGGACAAATGTCATTATGAGCTGCAACAGTTATTACTAACCTTATAAGTGC3’-(SEQ?ID?NO?189)5’TTTTACCCTACGGGCAGATGAGCCTGTGGGCTGCAACCGTTATTACTAACCTTATGAGCGC3’-(SEQ?ID?NO?190)5’TTTTACCATACGGACAAATGTCATTATGAGCTGCAACAGTTATTACTAACCTTATGAGTGC3’-(SEQ?ID?NO?191)5’TTTTACCTTGGGGACAAATGAGTTTTTGGGCTGCAACTGTTATTACTAATTTATTTTCTGC3’-(SEQ?ID?NO?192)5’TTCTTCCTTATGGGCAAATGTCTTTATGAGCTGCTACAGTTATTACTAACCTTATGAGTGC3’-(SEQ?ID?NO?193)5’TATTACCTTATGGACAAATGTCATTATGAGCAGCTACAGTTATTACTAATTTATTATCTGC3’-?(SEQ?ID?NO?194)5’TGCTTCCATACGGGCAAATGTCTCTGTGGGCTGCTACAGTAATTACTAATTTACTTTCTGC3’-(SEQ?ID?NO?195)5’TTTTACCTTATGGTCAAATGTCTTTATGAGCAGCTACAGTTATAACTAATTTAATGAGTGC3’(SEQ?ID?NO?196)

The present invention also extends to and shows the fungal DNA sequence that homology or sequence identity are arranged with the described dna sequence dna that contains described polymorphism, comprises the variation of the dna sequence dna of for example finding in the different samples of same species.These variations can for example cause owing to using alternative codon selection, change intron/exon plastosome group structure and aminoacid replacement.

The all or part of wild-type as herein described of encoding or the proteic dna sequence dna of mutant cell pigment b are preferably isolating form.For example, by from naturally occurring any material, carrying out partial purification.Described dna sequence dna is separable from (can derive from) or separate from (deriving from) fungi disclosed herein.

The present invention also provides the media of the computer-reader form of the described herein and claimed any described sequence of storage, described sequence comprises coding proteic all or part of dna sequence dna of mutant cell pigment b as herein described or protein sequence, and the existence of wherein said sudden change causes the resistance of fungi for any compound in strobilurins analogue or the same crossed resistance group; From the proteic all or part of dna sequence dna of encoding mutant type cytochrome b or the protein sequence that are selected from following fungi: it is mould that grape is given birth to single shaft, the mutation of causing a disease of standing grain powdery mildew wheat/barley, rye beak spore, the circle nuclear cavity bacteria, standing grain green-ball chamber bacterium, venturia inaequalis, Mycosphaerellafijiensis var.difformis, the monofilament shell, grape snag shell, standing grain is given birth to thorn dish spore, the melon and fruit corruption is mould, Colletotrichum gloeosporiodes, the tomato powder spore, Magnaporthe grisea, phytophthora infestans, Tartar's internal thread powdery mildew, the false downy mildew of Cuba, upright withered chain lattice spore, dry thread Pyrenomycetes, Mycosphaerellamusicola and Semen arachidis hypogaeae tail spore, wherein said protein is given the resistance of fungi for the compound in strobilurins analogue or the same crossed resistance group, and prerequisite is that described dna sequence dna or protein sequence are not the cytochrome b sequences of the little mushroom of newborn handle; Be selected from all or part of dna sequence dna or the protein sequence of the encoding wild type cytochrome b sequence of following a kind of fungi: it is mould that grape is given birth to single shaft, the mutation of causing a disease of standing grain powdery mildew wheat/barley, rye beak spore, the circle nuclear cavity bacteria, standing grain green-ball chamber bacterium, Mycosphaerella fijiensis var.difformis, the monofilament shell, grape snag shell, standing grain is given birth to thorn dish spore, the melon and fruit corruption is mould, Colletotrichum gloeosporiodes, the tomato powder spore, Tartar's internal thread powdery mildew, the false downy mildew of Cuba, upright withered chain lattice spore, dry thread Pyrenomycetes, Mycosphaerellamusicola and Semen arachidis hypogaeae tail spore; Or any allele specific oligonucleotide; Alleles-specific oligonucleotide probe disclosed herein, allele-specific primers, general primer or diagnostic primer.

Described computer readable medium can be used for for example homology search, mapping, haplotype typing (haplotyping), Geotype setting (genotyping) or any other analysis of biological information.Any computer readable medium all can use for example CD, tape, floppy disk, hard drive or computer chip.

Polynucleotide sequence of the present invention or its part, particularly relate to and identify that the single nucleotide polymorphism that this paper identifies (especially causes G in the coded albumen among fungal cell's pigment b ₁₄₃The G that A changes becomes C) those polynucleotide sequences, be valuable information source.By the described sequence information of storage in computer readable medium, use described information, the application of the easiest this information source of promotion with the standard biological information programme then.Polynucleotide sequence of the present invention is particularly useful as the integral part in the database, in order to carry out sequence identity and other searching analysis.Polynucleotide used herein and of the present invention or polynucleotide sequence relevant in the storage of sequence information described in the computer readable medium and the application in sequence library, comprise and to be reduced to, to change into tangible media or with any detectable chemical feature or the physical features of the polynucleotide of the present invention of tangible media storage, described tangible media is computer disks for example, is preferably computer-reader form.For example, chromatographic scan data or peak data, photographing scanning data or peak data, mass-spectrometric data, sequence gel (or other) data.

Also provide computer-based method in order to carry out Sequence Identification, said method comprising the steps of: a kind of polynucleotide sequence of computer readable medium is provided, and described polynucleotide sequence comprises a kind of polymorphism of the present invention; And the described polynucleotide sequence that contains polymorphism is compared with at least a other polynucleotide or peptide sequence,, promptly screen the existence of described polymorphism to identify identity (homology).

The present invention also provides and gives the fungal cell pigment b albumen of fungi for the resistance of the compound in strobilurins analogue or the same crossed resistance group, wherein in described protein, owing to existing sudden change to change normal glycine residue among the DNA of code for said proteins, described sudden change occur among the described DNA in the code for said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in the correspondence position of one or more bases on, prerequisite is that described sequence is not the cytochrome b sequence of the little mushroom of newborn handle.

In a preferred embodiment aspect this, the present invention also provides and gives the fungal cell pigment b albumen of fungi for the resistance of the compound in strobilurins analogue or the same crossed resistance group, wherein in described protein, owing to existing sudden change to change normal glycine residue among the DNA of code for said proteins, described sudden change occur among the described DNA in the code for said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in the correspondence position of second bit base on, prerequisite is that described sequence is not the cytochrome b sequence of the little mushroom of newborn handle.

The cytochrome b sequence of breast handle little mushroom is described (Eur.J.Biochem.235,54-63 (1996)) by Kraiczy etc., and described dna sequence dna is in the EMBL database, and the EMBL accession number is X87997.

Glycine residue in the protein aspect above-mentioned according to the present invention, preferably by an alternative aminoacid replacement, and described replacement causes described fungi to show resistance for any other compound in strobilurins analogue or the same crossed resistance group.

Sudden change according to the above-mentioned aspect of the present invention preferably causes described glycine residue to be selected from following a kind of aminoacid replacement: arginine, Serine, halfcystine, Xie Ansuan, aspartic acid, L-glutamic acid, preferably L-Ala.

On the one hand, the invention provides and to discern the proteic antibody of described mutant cell pigment b again.

Again on the one hand, the invention provides a kind of method, be used for detecting fungal cell's pigment b gene cause in coded albumen with brewing yeast cell pigment b residue 143 corresponding positions on the substituted sudden change of glycine residue, described method comprises: whether identifying suddenlys change described in the fungal nucleic acid sample exists, wherein any (or a kind of) mononucleotide polymorphic detection method based on come in described wild-type of own coding or the mutant protein with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in the sequence information of upstream and/or downstream about 30-90 Nucleotide of correspondence position of one or more bases.

In the preferred embodiment again aspect this, the invention provides a kind of method, be used for detecting fungal cell's pigment b gene and cause G in coded albumen ₁₄₃The sudden change that A replaces, described method comprises: whether identifying suddenlys change described in the fungal nucleic acid sample exists, wherein any (or a kind of) mononucleotide polymorphic detection method based on come in described wild-type of own coding or the mutant protein with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in the sequence information of upstream and/or downstream about 30-90 Nucleotide of correspondence position of second bit base.

In the preferred embodiment again aspect this, the invention provides a kind of method, be used for detecting fungal cell's pigment b gene and cause G in coded albumen ₁₄₃The guanine that A replaces becomes the sudden change of cytosine(Cyt), described method comprises: whether identifying suddenlys change described in the fungal nucleic acid sample exists, wherein any (or a kind of) mononucleotide polymorphic detection method based on come in described wild-type of own coding or the mutant protein with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in the sequence information of upstream and/or downstream about 30-90 Nucleotide of correspondence position of second bit base.

Preferably derive from according to the sequence information of the above-mentioned aspect of the present invention and to be selected from following a kind of fungi: it is mould that grape is given birth to single shaft, the mutation of causing a disease of standing grain powdery mildew wheat/barley, rye beak spore, the circle nuclear cavity bacteria, standing grain green-ball chamber bacterium, venturia inaequalis, Mycosphaerella fijiensis var.difformis, the monofilament shell, grape snag shell, standing grain is given birth to thorn dish spore, the melon and fruit corruption is mould, Colletotrichum gloeosporiodes, the tomato powder spore, Magnaporthe grisea, phytophthora infestans, Tartar's internal thread powdery mildew, the false downy mildew of Cuba, upright withered chain lattice spore, dry thread Pyrenomycetes, it is mould that Mycosphaerella musicola and Semen arachidis hypogaeae tail spore, described fungi more preferably are selected from the living single shaft of grape, the mutation of causing a disease of standing grain powdery mildew wheat/barley, rye beak spore, the circle nuclear cavity bacteria, standing grain green-ball chamber bacterium, Mycosphaerella fijiensis var.difformis, the monofilament shell, grape snag shell, standing grain is given birth to thorn dish spore, the melon and fruit corruption is mould, Colletotrichum gloeosporiodes, the tomato powder spore, Tartar's internal thread powdery mildew, the false downy mildew of Cuba, upright withered chain lattice spore, dry thread Pyrenomycetes, Mycosphaerella musicola and Semen arachidis hypogaeae tail spore.

About 30 of term used herein is meant that described sequence can comprise 30 Nucleotide at the most, and for example 5 to 10,15,20 or 25 Nucleotide maybe can comprise the Nucleotide more than 30.Of the present invention above-mentioned aspect, preferably used sequence information be in described wild-type of coding or the mutant protein with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in the upstream of correspondence position of second bit base and/or about 30 of downstream, the sequence information of 30 Nucleotide preferably.

According to nucleic acid of the present invention DNA preferably, the given the test agent of described nucleic acid be easily from fungal material total DNA prepared product, from fungal material or fungal material itself or contain the plant of fungal nucleic acid or the cDNA prepared product of seed extract.In the present invention, we described by with total DNA prepared product, cDNA prepared product and by directly with the fungal spore material as the template among the PCR, detection G ₁₄₃The A sudden change.People will appreciate that given the test agent can be the nucleotide sequence corresponding to sequence in the described given the test agent equally.That is to say, can at first isolate or, use it in the inventive method then with any technology easily all or part of described district in the pcr amplification sample nucleic acid for example.

The invention provides the method for suddenling change among the DNA that analyzes the farmland sample, described sudden change is compared with the analogue that comprises human sample because its origin is special, and it determines very different.The research of farmland sample is much more difficult, compare with only containing usually from the human sample of the DNA of body one by one, detect the catastrophic event that low frequency takes place at very a large amount of wild-type DNA and/or among other the organic irrelevant DNA that exists in from the field isolate, higher for the requirement of technology.

Can use any enzyme of polymerization easily, prerequisite is that it is not with the normal template sequence of discriminating of any significance degree and the capability between the mutant template sequence.The example of enzyme comprises the thermophilic enzyme that does not have remarkable 3 '-5 ' exonuclease activity easily, for example Taq archaeal dna polymerase, particularly ' Ampli Taq Gold ' ^TMOther suitable N-terminal disappearance modifier of archaeal dna polymerase (PE AppliedBiosystems), Stoffel fragment or Taq (Thermus aquaticus) or Tth (Thermusthermophilus) archaeal dna polymerase.

Again on the one hand, the invention provides can with the proteic all or part of fungal nucleic acid sequence bonded allele specific oligonucleotide of the encoding wild type cytochrome b that is selected from following fungi: it is mould that grape is given birth to single shaft, the mutation of causing a disease of standing grain powdery mildew wheat/barley, rye beak spore, the circle nuclear cavity bacteria, standing grain green-ball chamber bacterium, venturia inaequalis, Mycosphaerella fijiensis var.difformis, the monofilament shell, grape snag shell, standing grain is given birth to thorn dish spore, the melon and fruit corruption is mould, Colletotrichum gloeosporiodes, the tomato powder spore, Magnaporthe grise, phytophthora infestans, Tartar's internal thread powdery mildew, the false downy mildew of Cuba, upright withered chain lattice spore, dry thread Pyrenomycetes, Mycosphaerella musicola and Semen arachidis hypogaeae tail spore, wherein said oligonucleotide comprises one section sequence, the nucleotide sequence of the glycine residue on described recognition sequence coding and brewing yeast cell pigment b residue 143 corresponding positions.

In the preferred embodiment of the present invention aspect this, described fungal nucleic acid sequence is selected from that grape gives birth to that single shaft is mould, standing grain powdery mildew wheat/barley is caused a disease mutation, rye beak spore, circle nuclear cavity bacteria, standing grain green-ball chamber bacterium, Mycosphaerella fijiensis var.difformis, monofilament shell, grape snag shell, standing grain are given birth to thorn dish spore, the melon and fruit corruption is mould, Colletotrichum gloeosporiodes, tomato powder spore, Tartar's internal thread powdery mildew, the false downy mildew of Cuba, upright withered chain lattice spore, dry thread Pyrenomycetes, Mycosphaeyellamusicola and Semen arachidis hypogaeae tail spore.

According to the fungal nucleic acid of the above-mentioned aspect of the present invention DNA preferably.

In one side more of the present invention, we provide can with coding all or part of mutant cell pigment b proteic fungal nucleic acid sequence bonded allele specific oligonucleotide, wherein said oligonucleotide comprises one section sequence, described recognition sequence with brewing yeast cell pigment b residue 143 corresponding positions on coding be selected from following a kind of amino acid whose nucleotide sequence: arginine, Serine, halfcystine, Xie Ansuan, aspartic acid, L-glutamic acid, tryptophane, preferably L-Ala.

In the preferred embodiment of the present invention aspect this, we provide can with the coding all or part of mutant cell pigment b proteic fungal nucleic acid sequence bonded allele specific oligonucleotide that is selected from following fungi: it is mould that grape is given birth to single shaft, the mutation of causing a disease of standing grain powdery mildew wheat/barley, rye beak spore, the circle nuclear cavity bacteria, standing grain green-ball chamber bacterium, venturia inaequalis, Mycosphaerellafijiensis var.difformis, the monofilament shell, grape snag shell, standing grain is given birth to thorn dish spore, the melon and fruit corruption is mould, Colletotrichum gloeosporiodes, the tomato powder spore, Magnaporthe grisea, phytophthora infestans, Tartar's internal thread powdery mildew, the false downy mildew of Cuba, upright withered chain lattice spore, dry thread Pyrenomycetes, Mycosphaerellamusicola and Semen arachidis hypogaeae tail spore, wherein said oligonucleotide comprises one section sequence, described recognition sequence with brewing yeast cell pigment b residue 143 corresponding positions on coding be selected from following a kind of amino acid whose nucleotide sequence: arginine, Serine, halfcystine, Xie Ansuan, aspartic acid, L-glutamic acid, tryptophane, L-Ala preferably.

According to the fungal nucleic acid of the above-mentioned aspect of the present invention DNA preferably.

Again on the one hand, the invention provides a kind of alleles-specific oligonucleotide probe, described probe can detect in described DNA in the code for said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in fungal cell's pigment b gene pleiomorphism on the correspondence position of one or more bases.

In the preferred embodiment again aspect this, the invention provides a kind of alleles-specific oligonucleotide probe, described probe can detect in described DNA in the code for said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in fungal cell's pigment b gene pleiomorphism on the correspondence position of second bit base.

In the preferred embodiment again aspect the present invention is above-mentioned, described polymorphism is that guanine base becomes the cytosine(Cyt) base, and described sudden change is in being selected from following fungi: it is mould that grape is given birth to single shaft, the mutation of causing a disease of standing grain powdery mildew wheat/barley, rye beak spore, the circle nuclear cavity bacteria, standing grain green-ball chamber bacterium, venturia inaequalis, Mycosphaerella fijiensis var.difformis, the monofilament shell, grape snag shell, standing grain is given birth to thorn dish spore, the melon and fruit corruption is mould, Colletotrichum gloeosporiodes, the tomato powder spore, Magnaporthegrisea, phytophthora infestans, Tartar's internal thread powdery mildew, the false downy mildew of Cuba, upright withered chain lattice spore, dry thread Pyrenomycetes, Mycosphaerella musicola and Semen arachidis hypogaeae tail spore.

Preferably long 17-50 the Nucleotide of described alleles-specific oligonucleotide probe more preferably is about 17-35 Nucleotide, most preferably is about 17-30 Nucleotide.

The design of this class probe is conspicuous for common molecular biosciences scholar, and can be based on DNA or RNA sequence information.This class probe has any length easily, and for example 50 bases, 40 bases at the most at the most are length more easily is 30 bases at the most, for example long 8-25 or 8-15 base.Generally speaking, this class probe will comprise with described gene in corresponding wild type or the complete complementary base sequence of anomaly locus.Yet, if desired, can introduce one or more mispairing, prerequisite is the distinguishing ability of the described oligonucleotide probe of influence within reason.Probe of the present invention can have one or more marks, so that detect.

The present invention also provides nucleotide primer, and described nucleotide primer can detect according to nucleotide polymorphisms of the present invention.

According to another aspect of the present invention, allele-specific primers is provided, described primer can detect in described DNA in the code for said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in cytochrome b gene polymorphism on the correspondence position of one or more bases.

A preferred embodiment according to this aspect of the present invention, allele-specific primers is provided, described primer can detect in described DNA in the code for said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in cytochrome b gene polymorphism on the correspondence position of second bit base.

In aspect above-mentioned, the cytosine(Cyt) base is changed in the sudden change in the described dna sequence dna preferably guanine base.

More on the one hand, the invention provides the allele-specific primers that can detect the proteic fungal DNA sequence of encoding wild type cytochrome b that is selected from following a kind of fungi: it is mould that grape is given birth to single shaft, the mutation of causing a disease of standing grain powdery mildew wheat/barley, rye beak spore, the circle nuclear cavity bacteria, standing grain green-ball chamber bacterium, venturia inaequalis, Mycosphaerella fijiensis var.difformis, the monofilament shell, grape snag shell, standing grain is given birth to thorn dish spore, the melon and fruit corruption is mould, Colletotrichum gloeosporiodes, the tomato powder spore, Magnaporthe grisea, phytophthora infestans, Tartar's internal thread powdery mildew, the false downy mildew of Cuba, upright withered chain lattice spore, dry thread Pyrenomycetes, Mycosphaerella musicola and Semen arachidis hypogaeae tail spore, wherein said primer can detect with brewing yeast cell pigment b residue 143 corresponding positions on the coding glycine residue dna sequence dna.

In the preferred embodiment of the present invention aspect this, described fungal DNA sequence is selected from the dna sequence dna of following fungi: grape gives birth to that single shaft is mould, standing grain powdery mildew wheat/barley is caused a disease mutation, rye beak spore, circle nuclear cavity bacteria, standing grain green-ball chamber bacterium, Mycosphaerella fijiensis var.difformis, monofilament shell, grape snag shell, standing grain are given birth to thorn dish spore, the melon and fruit corruption is mould, Colletotrichum gloeosporiodes, tomato powder spore, Tartar's internal thread powdery mildew, the false downy mildew of Cuba, upright withered chain lattice spore, dry thread Pyrenomycetes, Mycosphaerella musicola and Semen arachidis hypogaeae tail spore.

In one side more of the present invention, we provide the allele-specific primers that can detect the proteic fungal DNA sequence of all or part of mutant cell pigment b of coding, wherein said allele-specific primers can detect with brewing yeast cell pigment b residue 143 corresponding positions on coding be selected from following a kind of amino acid whose dna sequence dna: arginine, Serine, halfcystine, Xie Ansuan, aspartic acid, L-glutamic acid, tryptophane, preferably L-Ala.

In the preferred embodiment of the present invention aspect this, we provide the allele-specific primers that can detect the proteic fungal DNA sequence of all or part of mutant cell pigment of the coding that is selected from following fungi b: it is mould that grape is given birth to single shaft, the mutation of causing a disease of standing grain powdery mildew wheat/barley, rye beak spore, the circle nuclear cavity bacteria, standing grain green-ball chamber bacterium, venturia inaequalis, Mycosphaerellafijiensis var.difformis, the monofilament shell, grape snag shell, standing grain is given birth to thorn dish spore, the melon and fruit corruption is mould, Colletotrichum gloeosporiodes, the tomato powder spore, Magnaporthe grisea, phytophthora infestans, Tartar's internal thread powdery mildew, the false downy mildew of Cuba, upright withered chain lattice spore, dry thread Pyrenomycetes, Mycosphaerellamusicola and Semen arachidis hypogaeae tail spore, wherein said primer can detect with brewing yeast cell pigment b residue 143 corresponding positions on coding be selected from following a kind of amino acid whose dna sequence dna: arginine, Serine, halfcystine, Xie Ansuan, aspartic acid, L-glutamic acid, tryptophane, preferably L-Ala.

Allele-specific primers usually is used from amplified reaction for example in the PCR reaction with shared primer one, and this provides discriminating between the allelotrope by the locational allelotrope of selective amplification particular sequence, for example is used for ARMS and measures.

We have designed now and have been used for above listed fungi strain G ₁₄₃The primer of A sudden change has shown that described primer can be used to detect reliably and intentinonally specific sudden change.Described primer detect in described DNA in the code for said proteins with yeast saccharomyces cerevisiae b residue 143 corresponding positions on amino acid whose triplet on the corresponding position of second bit base guanine base become the cytosine(Cyt) base.Described allele-specific primers is referred to herein as the diagnostic primer.Therefore, again on the one hand, the invention provides can with the template bonded diagnostic primer that comprises mutant fungal cell pigment b nucleotide sequence, the Nucleotide that last Nucleotide correspondence of wherein said primer 3 ' end exists in described mutant fungal cell's pigment b gene, and the existence of described Nucleotide causes the resistance of fungi for any other compound in strobilurins analogue or the same crossed resistance group.

Diagnostic primer of the present invention most preferably grow 26 Nucleotide, but this can a long 15-20 Nucleotide preferably to 20 Nucleotide of the youthful and the elderly.

In the preferred embodiment aspect the present invention is above-mentioned, the Nucleotide that exists in the correspondence position of penultimate Nucleotide (2) and wild-type cell pigment b sequence in the described primer is different.

In a preferred embodiment again, the Nucleotide that exists in the correspondence position of-3 Nucleotide of described primer and wild-type cell pigment b sequence is different just.

Other goes to stablize component and can mix with described-2 or-3 Nucleotide.

In the particularly preferred embodiment again aspect the present invention is above-mentioned, we provide can with the template bonded diagnostic primer that comprises mutant fungal cell pigment b nucleotide sequence, the Nucleotide that last Nucleotide correspondence of wherein said primer 3 ' end exists in described mutant fungal cell's pigment b gene, and wherein 10 at the most in all the other Nucleotide, for example 8,6,4,2,1 Nucleotide can change with respect to wild-type sequence at the most, and the characteristic of the described diagnostic primer of not remarkably influenced.

In the particularly preferred embodiment again aspect the present invention is above-mentioned, we provide the diagnostic primer that comprises following listed sequence and derivative thereof, wherein last Nucleotide of 3 ' end is identical with following listed sequence, and wherein 10 at the most in all the other Nucleotide, for example 8,6,4,2,1 Nucleotide can change at the most, and the characteristic of the described diagnostic primer of not remarkably influenced.

Be that the sequence of described diagnostic primer and the following institute sequence that provides are just the same easily.Preferably the ARMS primer in all aspects of the invention is grown 26 Nucleotide.Below great majority, in the listed primer, made penultimate Nucleotide different, so that described primer is gone to stablize, make it that described template is had more selectivity thus, and these primers have been particularly preferred according to the present invention with wild-type cytb sequence.For the technician in design of primers field, it is evident that under the situation that can influence described primer and required template bonded ability, the alternative base or the base except that above-mentioned base of above-mentioned base also can change sharply.

Primer #	Bacterial classification:	Be used to detect G 143The primer sequence of A (5 ' to 3 ')
			1	It is mould that grape is given birth to single shaft	CCTTGGTGACAAATGAGTTTTTGGAC ?(SEQ?ID?NO?22)
2	The mutation of causing a disease of standing grain powdery mildew wheat/barley	CCATACGGGCAGATGAGCCACTGGAC (SEQ?ID?NO?23)
			3	Rye beak spore	CCTTATGGACAGATGTCTTTATGATC (SEQ?ID?NO?24)
4	The circle nuclear cavity bacteria	CCCTACGGGCAAATGAGCCTTTGCGC (SEQ?ID?NO?25)
			5	Standing grain green-ball chamber bacterium	CCTTATGGTCAAATGTCTTTATGAAC (SEQ?ID?NO?26)
6	Mycosphaerella?fijiensis?var. difformis	CCTTATGGTCAAATGTCTTTATGATC (SEQ?ID?NO?27)
			7	The monofilament shell	CCCTTCGGTCAAATGTCGCTCTGGAC (SEQ?ID?NO?28)
8	Grape snag shell	CCCTACGGGCAGATGAGCCTATGGTC (SEQ?ID?NO?29)
			9	Standing grain is given birth to thorn dish spore	CCTTACGGACAAATGTCATTATGAAC

		(SEQ?ID?NO?30)
			10	The melon and fruit corruption is mould	CCTTGGTGTCAAATGAGTTTTTGGAC (SEQ?ID?NO?31)
11	Colletotrichum gloeosporiodes	CCTTATGGACAAATGTCATTATGAAC (SEQ?ID?NO?32)
			12	The tomato powder spore	CCCTACGGGCAGATGAGCCTGTGGAC (SEQ?ID?NO?33)
13	Tartar's internal thread powdery mildew	CCATACGGACAAATGTCATTATGAAC ?(SEQ?ID?NO?34)
			14	The false downy mildew of Cuba	CCTTGGGGACAAATGAGTTTTTGGAC ?(SEQ?ID?NO?35)
15	Upright withered chain lattice spore	CCTTATGGGCAAATGTCTTTATGAAC (SEQ?ID?NO?36)
			16	Semen arachidis hypogaeae tail spore	CCTTATGGACAAATGTCATTATGAAC (SEQ?ID?NO?37)
17	Dry thread Pyrenomycetes	CCATACGGGCAAATGTCTCTGTGGAC (SEQ?ID?NO?38)
			18	Venturia inaequalis	GTGTATGGTCAAATGAGCCTATGGCC (SEQ?ID?NO?39)
19	Magnaphorthe?grisea	CCTTATGGACAGATGTCATTATGAAC (SEQ?ID?NO?40)
			20	Phytophthora infestans	CCTTGGGGACAAATGAGTTTTTGGAC (SEQ?ID?NO?41)
21	Mycosphaerella?musicola	CCTTATGGTCAAATGTCTTTATGATC (SEQ?ID?NO?42)

Table 4: be used to detect G ₁₄₃The ARMS design of primers of A sudden change

As an example, included primer comprises in the table 4: ● be used for the ARMS primer of circle nuclear cavity bacteria and venturia inaequalis, they can be effective to or

Person's genomic dna prepared product or biological sample, described biological sample comprises the fungi branch

From thing, fungal cultures, fungal spore or infected vegetable material.● be used for monofilament shell, tomato powder spore, Tartar's internal thread powdery mildew, grape snag shell, the epidemic disease of causing a disease

The ARMS primer of mould, Magnaporthe grisea, dry thread Pyrenomycetes, they only can

To be effective to cDNA.● be used for that grape gives birth to that single shaft is mould, standing grain powdery mildew wheat or barley are caused a disease mutation, rye beak spore,

Standing grain green-ball chamber bacterium, M.fijiensis var.difformis, standing grain give birth to thorn dish spore, melon and fruit corruption mould,

Colletotrichum gloeosporiodes, the false downy mildew of Cuba, Semen arachidis hypogaeae tail spore, Mycosphaerella

The ARMS primer of musicolad and upright withered chain lattice spore, they can be effective to or base

Because of group DNA prepared product, cDNA prepared product, cDNA prepared product or biological sample

Product, described biological sample comprises fungal isolates, fungal cultures, fungal spore or is subjected to

The vegetable material that infects.

The cDNA material that does not wherein identify the bacterial classification of intron/exon group structure at present in purpose single nucleotide polymorphism (SNP) is on every side used in suggestion.

In order to make the primer that is shown in the table be suitable for standard A SPCR reaction, last base of 3 ' end should not stablized base and do not introduce corresponding to described point mutation.

This class primer can use any conventional synthetic method preparation.The example of these class methods can find in standard textbook, and described textbook for example is " Protocols ForOligonucleotides And Analogues:Synthesis And Properties; " MethodsIn Molecular Biology Series; The 20th volume; Sudhir Agrawal writes, HumanaISBN:0-89603-247-7; 1993; The 1st edition.

People will appreciate that, can design the extension of diagnostic primer, cause G in the coded protein with indication ₁₄₃The sudden change that A replaces does not exist.Preferably using the ARMS primer detects and causes G in the coded protein ₁₄₃The sudden change that A replaces does not exist.This paper has described and has been designed for this purpose primer.

The detection of wild-type sequence can be used as the contrast that detects described sudden change, also is necessary when needing the wild-type that exists in the quantitative assay sample and mutant allele.

Therefore more on the one hand, the invention provides can with the template bonded diagnostic primer that comprises wild-type fungal cell pigment b nucleotide sequence, the Nucleotide that wherein said primer 3 ' holds last Nucleotide correspondence to exist in wild-type fungal cell pigment b gene, and described wild-type fungi shows any other compound susceptibility in strobilurins analogue or the same crossed resistance group.

In the preferred embodiment of the present invention aspect this, the Nucleotide that exists in the penultimate Nucleotide (2) of described primer and the correspondence position of wild-type cell pigment b sequence is different.

In a preferred embodiment again, the Nucleotide that exists in-3 Nucleotide of described primer and the correspondence position of wild-type cell pigment b sequence is different.

Other goes to stablize component and can mix with described-2 or-3 Nucleotide.

Diagnostic primer of the present invention is most preferably grown 26 Nucleotide preferably to 20 amino acid of the youthful and the elderly, but can a long 15-20 Nucleotide.In the particularly preferred embodiment again aspect the present invention is above-mentioned, we provide can with the template bonded diagnostic primer that comprises wild-type fungal cell pigment b nucleotide sequence, the Nucleotide that last Nucleotide correspondence of wherein said primer 3 ' end exists in wild-type fungal cell pigment b, and wherein 10 at the most in all the other Nucleotide, for example 8,6,4,2,1 Nucleotide can change with respect to wild-type sequence at the most, and the characteristic of the described diagnostic primer of not remarkably influenced.

In the again particularly preferred embodiment of the present invention aspect this, we provide the diagnostic primer that comprises following listed sequence and derivative thereof, wherein last Nucleotide of 3 ' end is identical with following listed sequence, and wherein 10 at the most in all the other Nucleotide, for example 8,6,4,2,1 Nucleotide can change at the most, and the characteristic of the described diagnostic primer of not remarkably influenced.Be that the sequence of described diagnostic primer and the following institute sequence that provides are just the same easily.Below great majority, in the listed primer, made penultimate Nucleotide different,, made it that required template is had more selectivity thus so that described primer is gone to stablize with wild-type cell pigment b sequence.For the technician in design of primers field, it is evident that under the situation that can influence described primer and described template bonded ability, the alternative base or the base except that above-mentioned base of above-mentioned base also can change sharply.

Primer	Bacterial classification	Be used to detect the primer sequence (5 ' to 3 ') of WT sequence
			1	It is mould that grape is given birth to single shaft	CCTTGGTGACAAATGAGTTTTTGGAG(SEQ?ID?NO 43)
2	The mutation of causing a disease of standing grain powdery mildew wheat/barley	CCATACGGGCAGATGAGCCACTGGAG(SEQ?ID?NO 44)
			3	Rye beak spore	CCTTATGGACAGATGTCTTTATGATG(SEQ?ID?NO 45)
4	The circle nuclear cavity bacteria	CCCTACGGGCAAATGAGCCTTTGAAG(SEQ?ID?NO 46)
			5	Standing grain green-ball chamber bacterium	CCTTATGGTCAAATGTCTTTATGAAG(SEQ?ID?NO 47)
6	Mycosphaerella?fijiensis?var. difformis	CCTTATGGTCAAATGTCTTTATGATG(SEQ?ID?NO 48)
			7	The monofilament shell	CCCTTCGGTCAAATGTCGCTCTGGAG(SEQ?ID?NO

		49)
			8	Grape snag shell	CCCTACGGGCAGATGAGCCTATGGTG(SEQ?ID?NO 50)
9	Standing grain is given birth to thorn dish spore	CCTTACGGACAAATGTCATTATGAAG(SEQ?ID?NO 51)
			10	The melon and fruit corruption is mould	CCTTGGTGTCAAATGAGTTTTTGGAG(SEQ?ID?NO 52)
11	Colletotrichum gloeosporiodes	CCTTATGGACAAATGTCATTATGAAG(SEQ?ID?NO 53)
			12	The tomato powder spore	CCCTACGGGCAGATGAGCCTGTGGAG(SEQ?ID?NO 54)
13	Tartar's internal thread powdery mildew	CCATACGGACAAATGTCATTATGAAG(SEQ?ID?NO 55)
			14	The false downy mildew of Cuba	CCTTGGGGACAAATGAGTTTTTGGAG(SEQ?ID?NO 56)
15	Upright withered chain lattice spore	CCTTATGGGCAAATGTCTTTATGAAG(SEQ?ID?NO 57)
			16	Semen arachidis hypogaeae tail spore	CCTTATGGACAAATGTCATTATGAAG(SEQ?ID?NO 58)
17	Dry thread Pyrenomycetes	CCATACGGGCAAATGTCTCTGTGGAG(SEQ?ID?NO 59)
			18	Venturia inaequalis	GTGTATGGTCAAATGAGCCTATGGAG(SEQ?ID?NO 60)
19	Magnaporthe?grisea	CCTTATGGACAGATGTCATTATGAAG(SEQ?ID?NO 61)
			20	Phytophthora infestans	CCTTGGGGACAAATGAGTTTTTGGAG(SEQ?ID?NO 62)
21	Mycosphaerella?musicola	CCTTATGGTCAAATGTCTTTATGATG(SEQ?ID?NO 63)

Table 5: the ARMS design of primers that is used to detect wild-type sequence

In order to make primer shown in the table be suitable for standard A SPCR reaction, last base of 3 ' end should not stablized base and do not introduce corresponding to described wild-type sequence.

Above-mentioned example relates to the ARMS primer based on DNA forward chain.Special advantageous applications is based on the ARMS primer of DNA forward chain.

If desired, the ARMS primer also can be based on the reverse strand of DNA.According to the above-mentioned same principle that is used for the forward strand primer, design this class reverse strand primer, be that described primer can be to 20 amino acid of the youthful and the elderly, most preferably long 26 Nucleotide, but can a long 15-20 Nucleotide, and last Nucleotide of described primer 3 ' end and correlate template (being mutant or wild-type) coupling, preferably the penultimate residue is changed by the best, makes it and described correlate template not match.In addition, in described primer, in all the other Nucleotide 10 at the most, for example 8,6,4,2,1 Nucleotide can change at the most, and the characteristic of the described diagnostic primer of not remarkably influenced.

In many cases, be easily in one or more pcr amplification circulations, use the another kind of amplimer that diagnostic primer of the present invention and this paper are called general primer.In european patent number EP-B1-0332435, narrated aspect this one example easily.Described another kind of amplimer or forward or reverse general primer.For each bacterial classification, used primer is as follows.Primer shown below is a reverse primer.

	Bacterial classification	Primer sequence (5 ' to 3 ')
			1	It is mould that grape is given birth to single shaft	GATACCTAATGGATTATTTGAACCTACCT(SEQ?ID NO?64)
2	The mutation of causing a disease of standing grain powdery mildew wheat/barley	AACACCTAAAGGATTACCAGATCCTGCAC(SEQ?ID NO?65)
			3	Rye beak spore	TACACCTAAAGGATTACCTGACCCTGCAC(SEQ?ID NO?66)
4	The circle nuclear cavity bacteria	TTCAAGTACATCCAATTTCAATATACACT(SEQ?ID NO67)
			5	Standing grain green-ball chamber bacterium	TAACAGAAAATCCACCTCATACGAATTCAACTATG TCTTG(SEQ?ID?NO?68)
6	Mycosphaerella?fijiensis?var. difformis	AAACCTCCTCCAAATAAACTCAACTATATC(SEQ?ID NO?69)
			7	The monofilament shell	TAACTGAGAAACCCCCTCAGAGAAACTCCACAATA TCTTG(SEQ?ID?NO?70)
8	Grape snag shell	TTACAGAAAAACCACCTCAAAGAAACTCCACGATA

		TCTTG(SEQ?ID?NO?71)
			9	Standing grain is given birth to thorn dish spore	TAACTGAGAAACCTCCTCAAACGAATTCAACAATA TCTTG(SEQ?ID?NO?72)
10	The melon and fruit corruption is mould	CTACAGCAAATCCCCCCCATAACCAATCAACAATA TCTTT(SEQ?ID?NO?73)
			11	Colletotrichum gloeosporiodes	TAACAGAGAAACCTCCTCAAACGAATTCAACTATA TCTTG(SEQ?ID?NO?74)
12	The tomato powder spore	TTACAGAAAAACCTCCTCAAAGAAACTCCACGATA TCTTG(SEQ?ID?NO?75)
			13	Tartar's internal thread powdery mildew	TTACAGAGAAACCTCCTCAAATAAATTCAACTATA TCTTG(SEQ?ID?NO?76)
14	The false downy mildew of Cuba	CTACAGCAAAACCGCCCCACAACCAATCAACAATA TCTTT(SEQ?ID?NO?77)
			15	Upright withered chain lattice spore	TAACACTGAAACCTCCTCAAATGAACTCAACAATA TCTTG(SEQ?ID?NO?78)
16	Semen arachidis hypogaeae tail spore	AAACAGAGAAACCTCCTCATATAAATTCAACTAAA TCTTG(SEQ?ID?NO?79)
			17	Dry thread Pyrenomycetes	ACACGGAAAAGCCACCCCAGATTAACTCTACAAAA TCTTG(SEQ?ID?NO?80)
18	Venturia inaequalis	ATTGACTTAAGCCTCCCCACAGAAATTCGACTATA TCTTG(SEQ?ID?NO?81)
			19	Magnaporthe?grisea	TAAGAGAAAAACCACCTCAAATGAATTCAACAATA TCTTG(SEQ?ID?NO?82)
20	Phytophthora infestans	CAACAGCAAAACCTCCCCATAACCAATCAACAATA TCTTT(SEQ?ID?NO?83)
			21	Mycosphaerella?musicola	TAACAGAAAACCCACCTCAAATAAATTCAACTATA TCTTG(SEQ?ID?NO?84)

Table 6: the example of the general primer that uses with the ARMS primer

Under the situation of the longer sequence that is provided in table 6, the technician can use this information and design suitable primer.

It will be apparent for a person skilled in the art that described general primer can be the pathogen specific sequence that any identification easily is positioned at cytochrome b gene (or other goal gene) complementary strand of described sudden change selectivity primer 3 '.

The preferred long 50-400bp of size of described pcr amplification, but can long 30-2500bp maybe may long 30-10,000bp.

Can use a kind of primer that contrasts easily, described contrast design of primers is positioned at G ₁₄₃The upstream of A position.It will be apparent for a person skilled in the art that can right and wrong increase specifically any primer of described mutant nucleotide sequence or wild-type sequence of described contrast primer.When no matter whether having G when primer uses these primers with above-mentioned corresponding oppositely (" shared ") ₁₄₃The A point mutation all will be increased.

Primer	Bacterial classification	Contrast primer sequence (5 ' to 3 ')
			1	It is mould that grape is given birth to single shaft	GCCTTGGGGACAAATGAGTTTTTG(SEQ?ID NO?85)
2	The mutation of causing a disease of standing grain powdery mildew wheat/barley	GCCATACGGGCAGATGAGCCACTG(SEQ?ID NO?86)
			3	Rye beak spore	TCCTTATGGACAGATGTCTTTATG(SEQ?ID NO?87)
4	The circle nuclear cavity bacteria	ACCCTACGGGCAAATGAGCCTTTGAG(SEQ ID?NO?88)
			5	Standing grain green-ball chamber bacterium	TACCTTATGGTCAAATGTCTTTATGA(SEQ ID?NO?89)
6	Mycosphaerella?fijiensis?var. difformis	GTTTTACCTTATGGTCAAATGTCTTTATG (SEQ?ID?NO?90)
			7	The monofilament shell	TTCCCTTCGGTCAAATGTCGCTCTGG(SEQ ID?NO?91)
8	Grape snag shell	TACCCTACGGGCAGATGAGCCTATGG(SEQ ID?NO?92)
			9	Standing grain is given birth to thorn dish spore	TACCTTACGGACAAATGTCATTATGA(SEQ ID?NO?93)
10	The melon and fruit corruption is mould	TACCTTGGGGTCAAATGAGTTTTTGG(SEQ ID?NO?94)
			11	Colletotrichum gloeosporiodes	TACCTTATGGACAAATGTCATTATGA(SEQ ID?NO?95)
12	The tomato powder spore	TACCCTACGGGCAGATGAGCCTGTGG(SEQ ID?NO?96)
			13	Tartar's internal thread powdery mildew	TACCATACGGACAAATGTCATTATGA(SEQ ID?NO?97)
14	The false downy mildew of Cuba	TACCTTGGGGACAAATGAGTTTTTGG(SEQ ID?NO?98)

??15	Upright withered chain lattice spore	??TTCCTTATGGGCAAATGTCTTTATGA(SEQ ??ID?NO?99)
			??16	Semen arachidis hypogaeae tail spore	??TACCTTATGGACAAATGTCATTATGA(SEQ ??ID?NO?100)
??17	Dry thread Pyrenomycetes	??TTCCATACGGGCAAATGTCTCTGTGG(SEQ ??ID?NO?101)
			??18	Venturia inaequalis	??ACGTGTATGGTCAAATGAGCCTATGG(SEQ ??ID?NO?102)
??19	??Magnaporthe?grisea	??TACCTTATGGACAGATGTCATTATGA(SEQ ??ID?NO?103)
			??20	Phytophthora infestans	??TACCTTGGGGACAAATGAGTTTTTGG(SEQ ??ID?NO?104)
??21	??Mycosphaerella?musicola	??TACCTTATGGTCAAATGTCTTTATGA(SEQ ??ID?NO?105)

Table 7: the example of possible contrast design of primers

Can use several different methods to detect the diagnostic primer extension product and/or whether amplified production exists.These are conspicuous for the technician in nucleic acid detection method field.Preferable methods has been cancelled the needs of radio-labeling reagent.Particularly preferred detection method is based on the method whether fluoroscopic examination diagnostic primer extension product exists.Concrete detection method comprises gel electrophoresis analysis, " Scorpions " that describe among the PCT application number PCT/GB98/03521 with the name application of Zeneca Limited on November 25th, 1998 ^TMProduct detection method (content of described application is attached to herein by reference), " Taqman " ^TMThe method that the product detection method is for example described among patent No. US-A-5487972 and the US-A-5210015; The surface of " Molecular Beacons "  product detection method of summarizing in patent No. WO-95/13399 and general introduction in patent application WO 97/05280 strengthens the Raman resonance spectrum and learns (SERRS).Other preferred detection method comprises the PCR of the utilization ALEX that describes among the PCT application number WO 99/04037 of ARMS linear extension (ALEX) and announcement.Be to use in real time and detect easily." Scorpions " that describes among preferred especially the UK number of patent application GB2338301 with PCT application number PCT/GB98/03521 and announcement ^TMThe application of product detection method is used for all aspects of the invention described herein.Especially preferably the combination with ARMS described herein and Scorpion technology is used for all aspects of the present invention as herein described, and preferred detection method is based on the detection method of fluorescence.Many methods in these detection methods are suitable for using the quantitative examination of all above-mentioned primers.Can in different test tubes, carry out these different PCR, perhaps in a test tube, carry out these multiple different PCR.Use this class methods, can estimate the frequency that the point mutation molecule in the wild type molecule background exists.

Exemplify as this paper, we have used based on the ARMS primer of DNA forward chain and in conjunction with the Scorpion based on the DNA reverse strand and have detected as detection method.The Scorpion measuring element preferably comprises the reverse primer shown in the table 6.Those skilled in the art find out easily, also can use the alternative combinations of ARMS primer and Scorpion measuring element.For example, primer based on described DNA forward chain can be the combination of ARMS primer and Scorpion detection system, and it can be used with the general primer based on the DNA reverse strand, perhaps the primer based on the DNA reverse strand can be the combination of ARMS primer and Scorpion detection system, and it can be used with the general primer based on DNA forward chain.

In most of embodiment as herein described, described Scorpion measuring element is on described general primer.Described mutant nucleotide sequence and the specific ARMS primer of wild-type sequence and fluorescently-labeled described general primer are united use.These two are reflected in the different PCR test tubes and carry out, and when described probe combines with the amplicon that is produced, send fluorescence.Perhaps, described Scorpion element can be incorporated in the ARMS primer.In this case, these two kinds of ARMS primers can be with different fluorophore marks, and use with described general primer (at this moment being unlabelled).These three kinds of primers can be included in the same reaction, because the mutant amplicon of gained will cause sending different fluorescence with wild-type amplification.

As described in the UK number of patent application GB2338301 that announces, the Scorpions technology can be used with many different modes, for example: embed embodiment, wherein Scorpions primer afterbody has the intercalative dye between the base that can mix double chain acid molecule, and described intercalative dye becomes highly fluorescigenic when mixing; The FRET embodiment, it is right that the dyestuff that is wherein comprised in described primer forms transmission ofenergy; No quencher embodiment, wherein fluorophore is connected with Scorpions primer afterbody; The Bimolecular embodiment wherein can be introduced fluorophore and quencher two and separate but on the complementary molecule; Amplicon be caught and be surveyed to the capture probe embodiment wherein can specifically with identical non-amplification tail; With stem district (Stem) embodiment, wherein said primer afterbody comprises self complementary stem district.Described these embodiments in the UK number of patent application GB2338301 that announces, the content of described application is attached to herein by reference comprehensively.

The inventive method as herein described detects the sudden change of fungal gene reliably, the existence of wherein said sudden change causes fungi for the resistance of its target protein by the mycocide of chondriogen coding, its detection level scope is per 1,000,1 mutant allele of 000 wild-type allele is to per 10,1 mutant allele of 000 wild-type allele, preferable range is per 100,1 mutant allele of 000 wild-type allele is to 1 mutant allele of per 10,000 wild-type alleles.Method of the present invention also can detect the sudden change that takes place with upper frequency, 1 mutant allele of for example per 100 wild-type alleles, 1 mutant allele of per 10 wild-type alleles, or wherein only have mutant allele.Equally, method of the present invention can be used for detecting the frequency of wild-type allele in the mutant allele background.

The more combination of sensitive allele-specific primers extension make it become the technology of extremely valuable detection owing to utilizing ARMS technology and the quantitative method that is used for the present invention to make with the fungi sudden change of low frequency generation.

Detect the allelotrope that exists in the given isolate, make it possible to the result of phenotype bioanalysis and the DNA profile of target gene are interrelated.Simple point mutation has been explained the quantitative property of described resistance as the discovery of resistance mechanism, and the confirmation of single spore separation thing sequence, has confirmed described screening accuracy in resistance isolate and the susceptibility isolate frequency in measuring given the test agent.

With the exploitation that allele-specific primers extends, the specificity of ARMS and real-time fluorescence that this paper exemplifies detect the method that combines with the Scorpion system, make with the biological assay program in feasible method compare the existence that can analyze resistant mutation in the greater amount sample.Bigger sample number makes it possible to identify the resistant mutation that can detect low percentage frequency easily by biological assay to be lower than.This makes it possible to identify described resistance in described colony before obviously finding out resistance according to field data.With adopt biological assay can take a sample and compare with the position with the zone of testing, the high-throughput characteristic of this method make it possible to bigger zone and more the multi-section position take a sample and test.Detect the bonded allele-specific primers with real-time fluorescence and extend for example ARMS, permission is before observing the effect of described gene by biological assay according to phenotype in foreign cell and/or heterokaryocyte, detect the existence of resistant gene described in the colony, therefore reduced the error that when sample carries low frequency resistant gene type, sample is divided into susceptibility.With wait disease takes place in plant and compare, faster by reading (in real time) acquisition result simultaneously, this makes it possible to more apace the land for growing field crops situation to be done rapid reaction and the antagonism management is advised.

The explanation that one or more diagnostic primers of the present invention and the inventive method can be used and suitable packing are eligibly packaging together, and sell as test kit.Test kit will comprise one or more in following suitably: diagnostic, wild-type, contrast and shared Oligonucleolide primers: suitable Nucleotide triphosphoric acid, for example dATP, dCTP, dGTP, dTTP; A kind of suitable polysaccharase as previously described; With a kind of damping fluid.

More on the one hand, the invention provides a kind of method that detects the anti-mycocide resistance of phytopathogenic fungi, described method comprises the sudden change that detects in the fungal gene, the existence of wherein said sudden change causes the resistance of fungi to any other compound in strobilurins analogue or the same crossed resistance group, and described method comprises with any (or a kind of) mononucleotide polymorphic detection technique identifying to suddenly change described in the fungal nucleic acid whether exist.

In the embodiment again aspect this, the invention provides a kind of method that detects the anti-mycocide resistance of phytopathogenic fungi, described method comprises the existence of the amplicon that detection PCR produced between the reaction period, wherein said PCR reaction is included in suitable Nucleotide triphosphoric acid makes the given the test agent that comprises fungal nucleic acid contact with the diagnostic primer with a kind of being used under the existence of polymeric reagent, whether exist sudden change directly related in the detection of wherein said amplicon and the described nucleic acid, the existence of wherein said sudden change causes for the resistance of its target protein by the mycocide of chondriogen coding.

In a preferred embodiment aspect this, the invention provides a kind of phytopathogenic fungi that detects for the method for its target protein by the resistance of the mycocide of chondriogen coding, described method comprises: be used for making in the presence of the polymeric reagent given the test agent that comprises fungal nucleic acid to contact with the diagnostic primer of specific sudden change at suitable Nucleotide triphosphoric acid with a kind of, the existence of described sudden change causes the resistance for described mycocide, make that described diagnostic primer is extended when having described sudden change in described sample; Whether exist according to the diagnostic primer extension product then and detect described sudden change and whether exist.

In the preferred embodiment again aspect this, the invention provides a kind of phytopathogenic fungi that detects for the method for its target protein by the resistance of the mycocide of chondriogen coding, described method comprises: be used for making in the presence of the polymeric reagent given the test agent that comprises fungal nucleic acid to contact with the diagnostic primer of specific sudden change at suitable Nucleotide triphosphoric acid with a kind of, the existence of described sudden change causes the resistance for described mycocide, only make that when having described sudden change in described sample, described diagnostic primer is just extended; Whether exist according to the diagnostic primer extension product then and detect described sudden change and whether exist.

Aspect above-mentioned and the inventive method of describing in the embodiment, especially the existence that is suitable for suddenling change in the cytochrome b gene wherein causes the phytopathogenic fungi bacterial strain of mycocide resistance, the most especially be suitable for wherein said sudden change and cause phytopathogenic fungi bacterial strain for the resistance of the compound in strobilurins analogue or the same crossed resistance group, the most especially the described sudden change in described fungal DNA cause in coded albumen with brewing yeast cell pigment b residue 143 corresponding positions on the substituted situation of glycine residue under, more particularly cause G in the coded albumen in described sudden change ₁₄₃Under the situation that A replaces, especially described sudden change be with brewing yeast cell pigment b residue 143 corresponding positions on codon on second G become the sequence change of C.

More on the one hand, the invention provides a kind of for causing phytopathogenic fungi to the detection and the quantitative methods of its target protein by the mutation frequency of the resistance of the mycocide of chondriogen coding, whether described method comprises suddenling change in the detection fungal gene and exists, the existence of wherein said sudden change causes the resistance of fungi to described mycocide, and described method comprises with any (or a kind of) mononucleotide polymorphic detection technique identifying and quantitatively suddenly change described in the fungal nucleic acid whether exist.

In the preferred embodiment again aspect this, the invention provides a kind of for causing phytopathogenic fungi to the detection and the quantitative methods of its target protein by the mutation frequency of the resistance of the mycocide of chondriogen coding, whether described method comprises suddenling change in the detection fungal gene and exists, the existence of wherein said sudden change causes the resistance of fungi for any other compound in strobilurins analogue or the same crossed resistance group, and described method comprises with any (or a kind of) mononucleotide polymorphic detection technique identifying and quantitatively suddenly change described in the fungal nucleic acid whether exist.

In the embodiment again aspect this, the invention provides a kind of for causing phytopathogenic fungi to the detection and the quantitative methods of its target protein by the mutation frequency of the resistance of the mycocide of chondriogen coding, described method comprises: the existence of the amplicon that detection PCR produced between the reaction period, wherein said PCR reaction is included in suitable Nucleotide triphosphoric acid and exists the given the test agent that comprises fungal nucleic acid is contacted with suitable primer with a kind of polymeric reagent that is used for, whether exist described sudden change directly related in the detection of wherein said amplicon and the described nucleic acid, the existence of wherein said sudden change causes for the resistance of its target protein by the mycocide of chondriogen coding; Whether there is the relative existence that detects described sudden change according to the amplicon that produces during the PCR then or do not exist.

In a preferred embodiment aspect this, the invention provides a kind of for causing phytopathogenic fungi to the detection and the quantitative methods of its target protein by the mutation frequency of the resistance of the mycocide of chondriogen coding, described method comprises: be used for making in the presence of the polymeric reagent given the test agent that comprises fungal nucleic acid to contact with the diagnostic primer that does not have specific sudden change with the described nucleic acid existence of detection at suitable Nucleotide triphosphoric acid with a kind of, the existence of described sudden change causes the resistance for described mycocide, make when in described sample, having described suitable fungi template, exist with described specific sudden change and do not exist relevant described diagnostic primer to be extended; Whether there is the relative existence that detects described sudden change according to the diagnostic primer extension product then and do not exist.

In the preferred embodiment again aspect this, the invention provides a kind of for causing phytopathogenic fungi to the detection and the quantitative methods of its target protein by the mutation frequency of the resistance of the mycocide of chondriogen coding, described method comprises: be used for making in the presence of the polymeric reagent given the test agent that comprises fungal nucleic acid to contact with the diagnostic primer that does not have specific sudden change with the described nucleic acid existence of detection at suitable Nucleotide triphosphoric acid with a kind of, the existence of described sudden change causes the resistance for described mycocide, only make when in described sample, having described suitable fungi template, exist with described specific sudden change and do not exist relevant described diagnostic primer to be extended; Whether there is the relative existence that detects described sudden change according to the diagnostic primer extension product then and do not exist.

Aspect above-mentioned and the inventive method of describing in the embodiment, especially the existence that is suitable for suddenling change in the cytochrome b gene wherein causes the phytopathogenic fungi bacterial strain of mycocide resistance, the most especially be suitable for wherein said sudden change and cause phytopathogenic fungi bacterial strain for the resistance of the compound in strobilurins analogue or the same crossed resistance group, the most especially the described sudden change in described fungal DNA cause in coded albumen with brewing yeast cell pigment b residue 143 corresponding positions on the substituted situation of glycine residue under, more particularly cause G in the coded albumen in described sudden change ₁₄₃Under the situation that A replaces, especially described sudden change be with brewing yeast cell pigment b residue 143 corresponding positions on codon on second G become the sequence change of C.

Aspect another, the invention provides a kind of selection and be applied to the active fungicide of crop and the method for the suitableeest dispenser level thereof, described method comprises that analysis can infect the fungi sample of described crop, and detection and/or quantitative assay are from the existence of the sudden change in the gene of described fungi and/or do not exist, the existence of wherein said sudden change can cause selecting active fungicide and the suitableeest dispenser level thereof then for the resistance of its target protein by the mycocide of chondriogen coding.

In the particularly preferred embodiment of the present invention aspect this, described detection method comprises any (or a kind of) mononucleotide polymorphic detection technique, described detection method more preferably comprises: be used for making in the presence of the polymeric reagent given the test agent that comprises fungal nucleic acid to contact with the diagnostic primer of described specific sudden change at suitable Nucleotide triphosphoric acid with a kind of, make that described diagnostic primer is extended when having described sudden change in described sample; Whether exist according to the diagnostic primer extension product then and detect described sudden change and whether exist, and by being used for making in the presence of the polymeric reagent given the test agent that comprises fungal nucleic acid to contact with the diagnostic primer that does not have specific sudden change with a kind of with the described nucleic acid existence of detection at suitable Nucleotide triphosphoric acid, the existence of described sudden change causes for the resistance of its target protein by the mycocide of chondriogen coding, make when in described sample, having described suitable fungi template, exist with described specific sudden change and do not exist relevant described diagnostic primer to be extended; Whether exist according to the diagnostic primer extension product then and detect and the relative existence of quantitative described sudden change and not existing, reach quantitative.

In the again particularly preferred embodiment of the present invention aspect this, described detection method comprises: be used for making in the presence of the polymeric reagent given the test agent that comprises fungal nucleic acid to contact with the diagnostic primer of described specific sudden change at suitable Nucleotide triphosphoric acid with a kind of, only make that when having described sudden change in described sample, described diagnostic primer is just extended; Whether exist according to the diagnostic primer extension product then and detect described sudden change and whether exist, and by being used for making in the presence of the polymeric reagent given the test agent that comprises fungal nucleic acid to contact with the diagnostic primer that does not have specific sudden change with a kind of with the described nucleic acid existence of detection at suitable Nucleotide triphosphoric acid, the existence of described sudden change causes for the resistance of its target protein by the mycocide of chondriogen coding, only make when in described sample, having described suitable fungi template, exist with described specific sudden change and do not exist relevant described diagnostic primer just to be extended; Whether exist according to the diagnostic primer extension product then and detect and the relative existence of quantitative described sudden change and not existing, reach quantitative.

The inventive method of Miao Shuing existence of especially being suitable for suddenling change in the cytochrome b gene wherein herein causes the phytopathogenic fungi bacterial strain of mycocide resistance, the most especially be suitable for wherein said sudden change and cause phytopathogenic fungi bacterial strain for the resistance of the compound in strobilurins analogue or the same crossed resistance group, the most especially the described sudden change in described fungal DNA cause in coded albumen with brewing yeast cell pigment b residue 143 corresponding positions on the substituted situation of glycine residue under, more particularly cause G in the coded albumen in described sudden change ₁₄₃Under the situation that A replaces, especially described sudden change be with brewing yeast cell pigment b residue 143 corresponding positions on codon on second G become the sequence change of C.

Aspect another, the invention provides a kind of method of preventing and treating crop fungi infestation, described method comprises a kind of mycocide is applied to described crop that wherein described mycocide is selected in arbitrary system of selection according to the present invention described above.

The existence that above-mentioned the inventive method especially is suitable for suddenling change in the cytochrome b gene wherein causes the phytopathogenic fungi bacterial strain of mycocide resistance, the most especially is suitable for wherein said sudden change and causes phytopathogenic fungi bacterial strain for the resistance of the compound in strobilurins analogue or the same crossed resistance group.

More on the one hand, the invention provides the method that is used to detect Fungicidal active compound, described method comprises: cause for its target protein coming SCREENED COMPOUND by the fungal bacterial strain whether sudden change of the mycocide resistance of chondriogen coding exists at having tested according to the inventive method described herein; Measure Fungicidally active then at described fungal bacterial strain.

The existence that the inventive method described herein especially is suitable for suddenling change in the cytochrome b gene wherein causes the phytopathogenic fungi bacterial strain of mycocide resistance, the most especially be suitable for wherein said sudden change and cause phytopathogenic fungi bacterial strain for the resistance of the compound in strobilurins analogue or the same crossed resistance group, the most especially the described sudden change in described fungal DNA cause in coded albumen with brewing yeast cell pigment b residue 143 corresponding positions on the substituted situation of glycine residue under, more particularly cause G in the coded albumen in described sudden change ₁₄₃Under the situation that A replaces, especially described sudden change be with brewing yeast cell pigment b residue 143 corresponding positions on codon on second G become the sequence change of C.

By using the inventive method as herein described, can determine the suitable liquid application rate of the mycocide in described crop to be administered and/or suitable mycocide combination.

The inventive method as herein described is particularly suitable for monitoring in the crop fungi for the resistance of the compound in strobilurins analogue or the same crossed resistance group, described crop is cereal crop, fruits and vegetables for example, for example canola, Sunflower Receptacle, tobacco, beet, cotton, soybean, corn, wheat, barley, paddy rice, Chinese sorghum, tomato, mango, peach, apple, pears, strawberry, banana, muskmelon, potato, Radix Dauci Sativae, lettuce, Caulis et Folium Brassicae capitatae, onion, grape and lawn.

For example the sudden change of cytochrome b gene low and medium frequency is sensitive especially for detection line plastochondria encoding gene for the inventive method as herein described, make its approach that becomes a kind of phytopathogenic fungi of particularly useful commercially important screening generation mycocide resistance, wherein said resistance is because the sudden change in the plastosome encoding gene causes.

The accompanying drawing summary

Referring now to following non-limiting example and description of drawings the present invention, in the accompanying drawings:

Fig. 1 shows: the diagram that adopts the mould primer secondary structure of the living single shaft of Scorpion grape of MFold Zucker program.

Fig. 2 a shows: illustrate and adopt C Auele Specific Primer and Scorpion primer in wild-type DNA background grape to be given birth to the figure that the mould mutant DNA serial dilution of single shaft carries out pcr amplification (carrying out with double).As shown in the drawing, represent rhombus=1st line of 50%C/G, represent rhombus=7th line of 100%G, represent circle=2nd line of 10%C, represent circle=3rd line of 1%C, represent circle=4th line of 0.1%C, represent circle=5th line of 0.01%C, and represent circle=6th line of NTC.

Fig. 2 b shows: illustrate and adopt grape to give birth to the mould ARMS primer of single shaft carries out multiple experiment (carrying out with double) to 2 kinds of serial dilutions of mutant DNA in wild-type DNA background figure.As shown in the drawing, represent 1 by the line of rhombus representative: the result of 100Gfam, represented the result of 1: 500 Gfam to represent 1 by the line of circle representative by the line of trilateral representative: the result of 100Ctet, and represent 1 by the line of square representative: the result of 500Ctet.

Fig. 3 a shows: illustrate and adopt the figure of described three kinds of primers to the total DNA of standing grain powdery mildew (carrying out with double) of (specificity G/C and contrast primer) amplification.As shown in the drawing, represent to represent the result of 1/100 standard the result of 1/100Gmix by the line of trilateral representative, and represent the result of 1/100Cmix by the line of fork-shaped symbology by the line of rhombus representative.

Fig. 3 b shows: illustrate and adopt the figure of described three kinds of primers to amplification responsive type standing grain powdery mildew isolate.As shown in the drawing, represent Qmix, 1/100 result=1st line by the line of rhombus representative, line by square representative is represented Qmix, 1/1000 result=2nd line is represented Gmix, 1/100 result=3rd line by the line of trilateral representative, line by the circle representative is represented Gmix, 1/1000 result=4th line is represented Cmix by the line of fork-shaped symbology, 1/100 result=5th line, line by the rhombus representative is represented Cmix, 1/1000 result=6th line.

Fig. 4 shows: illustrate and adopt the figure of described three kinds of primers to amplification resistance standing grain powdery mildew isolate.As shown in the drawing, represent Qmix, 1/100 result=1st line by the line of rhombus representative, line by square representative is represented Qmix, 1/1000 result=2nd line is represented Gmix, 1/100 result=3rd line by the line of trilateral representative, line by the rhombus representative is represented Gmix, 1/1000 result=4th line is represented Cmix by the line of fork-shaped symbology, 1/100 result=5th line, line by the circle representative is represented Cmix, 1/1000 result=6th line.

Fig. 5 a shows: illustrate and adopt the figure of wild-type Auele Specific Primer to the pcr amplification (carrying out with double) of the wild-type rye beak sporonin grain serial dilution of amplification.As shown in the drawing, represent the result of G plasmid 10^8 by the line of rhombus representative, represent to represent the result of G plasmid 10^6 the result of G plasmid 10^4 by the line of square representative, and represent the result of G plasmid 10^2 by the line of circle representative by the line of trilateral representative.

Fig. 5 b shows: illustrate and adopt the figure of wild-type Auele Specific Primer to the pcr amplification (carrying out with double) of the wild-type of the maximum concentration that increases and mutant rye beak sporonin grain.Can observe from this figure, represent the result of G plasmid 10^8, and represent the result of c plasmid 10^8 by the line of trilateral representative by the line of rhombus representative.

Fig. 6 a shows: illustrate and adopt the figure of mutant Auele Specific Primer to the pcr amplification (carrying out with double) of the mutant rye beak sporonin grain serial dilution of amplification.As shown in the drawing, represent the result of c plasmid 10^8 by the line of rhombus representative, represent to represent the result of c plasmid 10^6 the result of c plasmid 10^4 by the line of square representative, and represent the result of c plasmid 10^2 by the line of circle representative by the line of trilateral representative.

Fig. 6 b shows: illustrate and adopt the figure of mutant Auele Specific Primer to the pcr amplification (carrying out with double) of the wild-type of the maximum concentration that increases and mutant rye beak sporonin grain.Can observe from this figure, represent the result of G plasmid 10^8, and represent the result of c plasmid 10^8 by the line of trilateral representative by the line of rhombus representative.

Fig. 7 a, 7b and 7c show: illustrate employing G primer in three kinds of diluents through the figure of pcr amplification rye beak spore DNA and cDNA template (carrying out) with double.Can observe from this figure, represent total DNA by the line of rhombus representative, and represent cDNA by the line of trilateral representative.

Fig. 8 a, 8b and 8c show: illustrate employing C primer in three kinds of diluents through the figure of pcr amplification rye beak spore DNA and cDNA template (carrying out) with double.Can observe from this figure, represent total DNA by the line of rhombus representative, and represent cDNA by the line of trilateral representative.

Fig. 9 a and 9b show: illustrate and adopt the figure of described three kinds of primers to amplification circle nuclear cavity bacteria P13 and P15 strain isolated (carrying out with double) in two kinds of diluents.As shown in the drawing, Fig. 9 a represents rhombus=1st line of S-mix, represent trilateral=2nd line of S-mix 1/10, represent square=3rd line of G-mix, represent circle=4th line of G-mix 1/10, represent circle=5th line of C-mix, represent square=6th line of C-mix 1/10.In Fig. 9 b, represent rhombus=1st line of s-mix, represent s-mix, 1/10 trilateral=2nd line, represent square=3rd line of g-mix, represent g-mix, 1/10 circle=4th line, represent circle=5th line of c-mix, represent c-mix, square=6th line of 1/10.

Detailed Description Of The Invention

Embodiment:

In following preceding four embodiment, with Scorpion ^TMSystem (AstraZenecaDiagnostics) is as the product detection system.This detection system has been carried out comprehensive description on November 25th, 1998 among the PCT application number PCT/GB98/03521 with the application of Zenaca Limited name, and the content of described application is attached to herein by reference.This novel detection system is used a kind of tailed primer and a kind of integrated signal system.The tail that described primer has a template land and comprises a joint and a target land.When using, the complementary sequence hybridization in the target land in the described tail and the extension products of described primer.With this target-specific hybrid incident and a kind of signalling system coupling, wherein hybridization causes detectable variation.The detection method of this system provides many significant advantages that are better than other system.Only need a kind of single primer/detector kind.This provides simplicity and enhanced specificity based on the increase of the described target land availability that is used for hybridizing with described primer extension product.Described new synthetic primer extension product is described target kind, and therefore available output signal is directly related with the primer amount of being extended.This does not rely on extra hybridisation events or enzymatic step.Because for competition is limited with interchain in the chain of described probe site, so simplified the design of probe.Because described interaction is monomolecular, so signal reaction is very quick, provide the cycle rate that improves as the conventional efficient key property.

She Ji Scorpion primer has following common modification in the following embodiments: ● hexaethylene glycol (HEG) monomer, as blocking part, between the template land and described tail region of described primer, this part prevents the chain copy of polymerase-mediated described primer template tail region.● a kind of FAM fluorescence molecule is added 5 ' end of described primer.FAM is a kind of fluorescence molecule, and it can be for example easily by the 488nm laser detection of ABI PRISM 7700 instruments (PE Biosystems).● MR is the non-fluorescent quenching agent that is connected in the uridylic residue

Other fluorescence molecule and cancellation mechanism also go for the Scorpion design of primers, also will be applicable to the present invention.

In embodiment 4, also intercalative dye is used as a kind of testing mechanism.Described 18 embodiment combine, described evaluation, and embodiment 1,2 and 6 has been described also by the evaluation from the part cytochrome b gene order of the fungal isolates of other compound in anti-strobilurins analogue and the same crossed resistance group from the part cytochrome b gene order of one group of important wild-type fungal isolates.Described method is specially adapted to strobilurins analogue azoxystrobin and picoxystrobin.

Embodiment 1

In embodiment 1, we described the part grape give birth to the evaluation of the mould cytochrome b gene sequence of single shaft, cause grape give birth to single shaft mould in strobilurins analogue resistance single nucleotide polymorphism (SNP) evaluation and be used for monitoring grape and give birth to the confirmation that the PCR in real time Scorpion of mould this SNP of single shaft measures.Also described and carried out multipleization (mulplexing) method that PCR in real time is measured.It is the pathogenic agent of downy mildew of garpe that grape is given birth to single shaft mould.

Grape is given birth to the mould wild-type ES2B strobilurins analogue responsive type strain isolated of single shaft and was collected in Spain in 1996.This strain isolated is not exposed to the strobilurins analogue as yet and selects.Manually choose infected grape leaf, it is stored in the polyethylene bag, and deliver to Jealotts ' s Hill Research Station (Zeneca Agrochemicals).During arrival, blade is placed on the moist blotter in the plastics casing in pairs, and will carries on the back an axle sporulation face and put together, cultivated 24-48 hour at 21-24 ℃.Downcut single scab from blade, sporocyst is suspended in the 5ml deionized water, be sprayed onto then on the abaxial surface of 5-6 week grape seedling in age (Ohanez mutation (var.Ohanez)).The plant of inoculation was newly cultivated 24 hours in (humidity chamber) (surrounding temperature, relative humidity 100%) in the dampening chamber, moved to (the daytime 24 ℃/r.h.60% of growth room then; Night 18 ℃/r.h.95%; Day is long 16 hours; 6,000 lux).After 6 days plant is retracted the dampening chamber and reach 24 hours again, after this successful infection shows as the sporulation scab on the back of the body axle blade face.The cultivation of further going down to posterity as mentioned above transfers to 5 with sporangia suspension, 000-10,000 sporocyst/ml.

Use based on big male epidemic disease mould, the primer of (Phytophthora megasperma) and Aspergillus nidulans (Aspergillus nidulans) cytochrome b gene conserved regions (Cytb12F (5 ' TGAACATATTATGAGAGATGT3 ') (SEQ ID NO 106) and Cyt10R (5 ' AATTGCATAAAAAGGTAAAAA3 ') (SEQ ID NO 107), they have described the profile based on the sequence of the amino acid district 66 of the encode true mycetocyte pigment b of yeast saccharomyces cerevisiae numbering system and 281), amplification coding wild-type grape is given birth to quite most dna fragmentation in the mould cytochrome b sequence of single shaft.Use the phenol/chloroform extraction scheme, from strobilurins analogue responsive type strain isolated, extract DNA.At 30ml redistilled water (ddH ₂O) in washing from disease cover 90-100% 6 blades (derive from artificial inoculation 6 age in week grape seedling) sporocyst.Sporangia suspension is filtered by Miracloth (Calbiochem catalog number 475855), then in 4 ℃ with 3600rpm centrifugal 10 minutes.Then that sporocyst is freezing in liquid nitrogen, grind to form fine powder with the pestle and the mortar that clean in advance and sterilize by acid elution and autoclaving.Add 0.5ml lysis buffer (200mM Tris-HCl (pH 8.5), 250mMNaCl, 25mM EDTA and 0.5%SDS), and suspension is transferred in the aseptic screw socket Eppendorf of the 1.5ml pipe.Add 0.5ml phenol/chloroform/primary isoamyl alcohol (25: 24: 1) mixture immediately, mix for several times by reversing then.With described pipe centrifugal 30 minutes, water is transferred in the new Eppendorf pipe then with 14000rpm.Repeat phenol/chloroform/isoamyl alcohol extracting then, but specifically with described pipe with 14000rpm only centrifugal 15 minutes.After in water being transferred to new Eppendorf pipe, carry out last chloroform extraction.By the 3M sodium acetate (pH 5.5) of adding 0.1 volume and the Virahol of 0.6 volume, precipitate fungal DNA then.After reversing for several times, with described pipe in 4 ℃ with 14000rpm centrifugal 20 minutes.Use 70% ethanol with DNA washing of precipitate 2 times then, vacuum-drying, and be resuspended in 50 μ l ddH ₂Among the O.Check DNA output and quality by gel electrophoresis, in redistilled water, prepare serial dilution (1: 10,1: 100 and 1: 1000) as the mould material among the PCR subsequently.According to the manufacturer's of Taq polysaccharase (Gibco) suggestion, set up PCR, the end reaction volume is 100ul.Described primer is added in the reaction, to final concentration be 1pmole/ μ l.Every kind of DNA diluent 10 μ l are added among the suitable PCR.Carry out standard program, with the danger of restriction PCR pollution.Carry out 30 circulations on Hybaid Omn-EPCR instrument, each circulation is 94 ℃ 45 seconds, 42 ℃ 45 seconds and 72 ℃ 1 minute 30 seconds.Also carry out 94 ℃ of initial step of 3 minutes and 72 ℃ of last extensions of 10 minutes.By gel electrophoresis analysis 18 μ l reaction mixtures, assess the efficient of PCR then.Then the sample of the described about 500bp PCR product of 2 μ l is cloned in TAPCR clone's pCR2.1 carrier (Invitrogen catalog number K4500-01), and is transformed into intestinal bacteria (Escherichia coli) cell (TOP10 OneShot according to manufacturer's suggestion ^TMCompetent cell) in.By carrying out Wizard minipreps (Promega catalog number A7100) according to manufacturer's explanation and analyzing the restriction digest, check whether a series of institutes DCRP exists the insertion fragment with EcoRI.Then with M13 forward primer and reverse primer to having 6 cloning and sequencings (ABI377XL automatic sequencer) of suitable insertion fragment (about 500bp).When using associated biomolecule information software (Seqman, when Editseq and Macaw) analyzing nucleotides sequence column data from these researchs, find a kind of new cytochrome b gene that has close homology with the mould cytochrome b sequence of for example big male epidemic disease of other known oomycetes of new sequence encoding of gained.In table 3, can find coding G ₁₄₃The gained grape of the second bit base upstream 30bp and downstream 30bp is given birth to the sequence of mould 61 the nucleotide sequence sections of single shaft in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).

Based on above-mentioned analysis be used for increase subsequently that to give birth to the mould Auele Specific Primer of single shaft be that (5 ' TCTTAAAATTGCATAAAAAGG3 ' (SEQID NO 109), described primer have described the profile according to the amino acid district 73-283 of fungal cell's pigment b of yeast saccharomyces cerevisiae numbering system for PLAS17F (5 ' AAATAACGGTTGGTTAATTCG3 ') (SEQID NO 108) and PLAS15R for the grape in purpose cytochrome b district.

Identify a grape at a test position and give birth to the mould strobilurins analogue of single shaft resistance strain isolated.Collect infected grape leave, and process as mentioned above basically, still sample being gone down to posterity to cultivate is large-scale colony (mass population), and does not separate with single scab before test.The testing method that confirms strobilurins analogue resistance is to carry out preventative sprinkling in 24 hours on the grape seedling in 4 ages in week.By with 5mg strobilurins analogue (used strobilurins analogue refers to azoxystrobin among all embodiment of this paper) (technologic material, purity 97%) be dissolved in the 1ml acetone, and in deionized water, further diluting under the room temperature, obtain 10ppm spray rate (dosage of known generation 100% control strobilurins analogue responsive type baseline separation strain), preparation chemical dilution series.The abaxial surface that sprays the target blade with the DeVilbiss spray gun with 10psi keeps to maximum.Control plant only spends deionized water spray.Allow treated plant dried overnight in growth room (condition as mentioned above).Carry out the inoculation that grape is given birth to the mould given the test agent of single shaft with 5,000 sporocysts/ml, the plant of the new inoculation of cultivation as discussed previously then.Inoculate back 7 days, isolate any potential resistance grower on treated blade,, be used for pcr analysis so that enough materials to be provided with its cultivation of going down to posterity.This strain isolated called after T5.

With PLAS17F and the PLAS15R primer part cytochrome b gene order that from described resistance strain isolated (T5), increases.With above-mentioned phenol/chloroform extraction scheme, from this strain isolated, extract total genomic dna (nuclear DNA and Mitochondrial DNA).Check existence and the quality of DNA once more by gel electrophoresis, in redistilled water, diluted suitable aliquot sample and be used for PCR research with 1: 10 and 1: 100.Then every kind of DNA diluent of 10 μ l is added Ready.To.Go ^TMIn the Taq polysaccharase PCR microballon (Amersham Pharmacia Biotech production number 27-9555-01), make 25 μ l with PLAS17F and PLAS15R primer solution, every kind to primer final concentration 1pmole/ μ l.Carry out standard program, with the danger of restriction PCR pollution.Carry out 30 round-robin PCR then on HybaidOmn-E PCR instrument, each circulation is 94 ℃ 45 seconds, 52 ℃ 45 seconds and 72 ℃ 1 minute 30 seconds.Also comprise an initial step-94 ℃ 3 minutes and last extension step-72 ℃ 10 minutes.These PCR carry out with double.After passing through the described PCR of gel electrophoresis analysis 10 μ l on the 0.8%TBE sepharose, before the clone, merge about 500bp PCR product of gained.Then the PCR product sample of the described merging of 1 μ l is cloned in TA clone's pCR2.1 carrier (Invitrogen), and is transformed into Bacillus coli cells (TOP10 One Shot according to manufacturer's suggestion ^TMCompetent cell) in.By carrying out Wizard minipreps (according to the explanation of Promega) and analyzing the restriction digest, check whether a series of institutes DCRP exists the insertion fragment with EcoRI.Insert 10 cloning and sequencings (ABI377XL automatic sequencer) of fragment (about 500bp) with M13 forward primer and reverse primer to having correct size then.

Under all 10 kinds of situations, analyze described sequence data with suitable bioinformation software (Seqman, Editseq andMacaw software), when comparing with wild-type sequence, the point mutation that discloses a G--＞C in the described cytochrome b sequence.This DNA point mutation causes that the last glycine of 143 (according to yeast saccharomyces cerevisiae amino acid coding schemes) becomes L-Ala in the position.

Design different specificity ARMS grapes and give birth to the mould primer of single shaft, to detect this G ₁₄₃Whether the A point mutation exists:

Two kinds of forward ARMS primer: G-sp-f-1:CCTTGGTGACAAATGAGTTTTTGTGG (SEQ ID NO 110) G-sp-f-2:CCTTGGTGACAAATGAGTTTTTGGAG (SEQ ID NO 111) based on wild-type sequence

Based on G ₁₄₃Two kinds of forward ARMS primer: C-sp-f-1:CCTTGGTGACAAATGAGTTTTTGGCC (SEQ ID NO 112) C-sp-f-2:CCTTGGTGACAAATGAGTTTTTGGAC (SEQ ID NO 113) of A sudden change

Be positioned at the contrast primer of described point mutation upstream: STAND:GCCTTGGGGACAAATGAGTTTTTG (SEQ ID NO 114) with design

In all above-mentioned ARMS primers ,-1 base (3 ' end base of described primer sequence) is corresponding to described target spot mutational site.The base that shows with runic is different from the base that the wild-type grape is given birth to the mould cytochrome b sequence of single shaft.In all described ARMS primers (not being the contrast primer) ,-20 bases become the T base from the G base.Carrying out this change is in order to destroy mix (run) of G base.In G-sp-f-2 and C-sp-f-2 primer ,-2 become the A base from the G base.In G-sp-f-1 ,-3 become the T base from the G base.In the C-sp-f-1 primer, described-2 primers become the C base from the G base.Carry out these sequences and change, so that described primer is gone to stablize, and make any primer extension have more specificity for corresponding template.Embodiment in the described document shows, the ARMS primer is gone to stablize, and has reduced the danger (Newton etc., Nucleic Acid Research 17 (7) 2503-2516 1989) of primer wrong start (mispriming) on wrong template.

Scorpion ^TMThe product detection system as testing mechanism, is mixed this detection system on the reverse primer in this case.The amplicon that is produced is with described ARMS primer duration 234bp, with contrast primer duration 235bp.With Oligo 5 and MFold program (MFold energy minimization method (Zucker, M. (1989) Science 244, the 48-52 of Zucker; SantaLucia, J.Jr (1998) .Proc.Natl.Acad.Sci.USA 95,1460-1465) secondary structure is fitted in the Asia that accommodates most of prediction RNA or dna molecular) design Scorpion primer.The sequence that grape is given birth to the mould Scorpion primer of single shaft is: 5 ' FAM- CCCGCCGTAATTGTAGGGGCTGTACTAATAC GGCGGGThe zone of (SEQ ID NO 115) MR-HEG-GATACCTAATGGATTATTTGAACCTACCT3 ' (SEQ ID NO 116) underscore is that hair clip forms part, FAM is a fluorescein(e) dye, MR is the non-fluorescent quenching agent that is connected in the uridylic residue, and HEG is the hexaethylene glycol monomer that blocking-up is duplicated.The sequence that italic is represented is the reverse primer sequence, and the sequence that runic is represented is the extension products bonded Scorpion sequence with described reverse primer.

The stem ring secondary structure (referring to Fig. 1) that can manifest this Scorpion primer with the MFold program.The energy that prediction has in its inertia form is-2.2 kcal/mol.Yet when extension products existed, described hairpin structure was separated, because the probe sequence of Scorpion primer combines with extension products, the prediction energy is-6.1 kcal/mol.This separates the FAM dyestuff with its quencher, for example cause can detect fluorescent emission with ABI Prism 7700 instruments.Therefore, compare, be annealed on the new synthetic chain helping the Scorpion element on the energetics with the stem ring configuration in the Scorpion inert condition.

(Lab 5005, and Medical andBiological Sciences Building is Southampton) synthetic by Oswel DNA Service for all primers.Before using, every kind of described primer in being the redistilled water of 500 μ l, cumulative volume is diluted to 5 μ M respectively.In PCR, primer further is diluted to the final concentration of 500nM then.

In all cases, the AmpliTaq Gold enzyme (Perkin-Elmer/ABI) that in reaction mixture, all comprises 1 unit/25ul reactant.Reaction mixture also contains 1 * damping fluid (10mM Tris-HCl (pH 8.3), 50mM KCl, 3.5mM MgCl ₂, 0.01% gelatin) and 100 μ M dNTP.Amplification is carried out in ABI Prism 7700 instruments, to carry out the successive fluorescence monitoring.After 95 ℃ 10 minutes one initial circulation, carry out 50 circulations: 95 ℃ of 15 seconds and 60 ℃ 45 seconds.At all round-robin annealing/extension stage monitoring fluorescence.

At first by with the plasmid DNA of various different concns as template, verify that primer is to be used for this alanysis.Carry out this checking and be specificity and sensitivity in order to check described design of primers.With part wild-type cell pigment b gene order with contain G accordingly ₁₄₃The tract of A sudden change is cloned in the TA pCR2.1 carrier (Invitrogen) and is used for this verification method.Compare with the G-sp-1 primer with C-sp-f-1, preferred C-sp-f-2 and G-sp-f-2 primer obtain more consistent result because repeat PCR, and specificity is slightly strong.In all cases, plasmid DNA is diluted in 1mg/ml bovine serum albumin (BSA).

Figure shown in Fig. 2 a has described the PCR with the mutant plasmid diluent in the ARMS C-sp-f-2 primer amplification wild-type template background.Because C plasmid extent of dilution reduces, therefore postponed fluoroscopic examination in the wild plasmid background.In all cases, the plasmid final concentration in described PCR is 1 * 10 ⁷Molecule/μ l.With every kind of 10 times of diluents of C plasmid, in fluoroscopic examination, postpone 3-4 circulation.When only there is 1 when copy in the C plasmid in 10000 copies in the wild plasmid background, still available described specificity ARMS primer detects.C-sp-f-2 primer integral body has specificity to its template corresponding, because can not detect fluorescence in 100% wild plasmid sample in this experiment.

Studied and can be used as the different possible material that starting material are used to use the resistance monitoring of this Scorpion technology to measure.In this research, use three kinds of different strain isolateds.For every kind of these isolate, both directly collect sporocyst from field pathology grape leave, also the blade (as previously mentioned) of directly growing from the room temperature of artificial inoculation is collected sporocyst.In both cases, all wash sporocyst from blade with redistilled water.Then by centrifugal collection sporocyst, and remain in-80 ℃ stand-by.Then the sporocyst sample is resuspended in the 1ml redistilled water, and in redistilled water, diluted once more with 1: 10 and 1: 100.Carry out PCR in real time as mentioned above and measure, just also in each PCR, add bovine serum albumin (0.25 μ g/ μ l), and with every kind of diluent of 5 μ l as template.Every kind of dilution is carried out with double, adds C-sp-f-2, G-sp-f-2 and contrast forward primer with shared Scorpion reverse primer respectively.Obtain good result with the sporocyst of collecting from the grape leaf of greenhouse growth as starting material, provide consistent cycle threshold (Ct).With the sporocyst of directly collecting from the field grape leaf obtain poor as a result, therefore according to following two kinds of methods, to improve the quality of data: (compare with the blade of greenhouse growth with the restraining effect that reduces among the PCR, the field material will contain many more possible pollutents), and make described DNA more can be used for amplification.

In order to reduce the influence of constituents for suppressing, improve the BSA concentration that adds in the reactant, and washing agent Tween-20 is added among the PCR PCR.In order to make described DNA more can be used for amplification, carry out an initial PCR, with the PCR product that derives from this reaction as the template in the PCR in real time, the spore diluent boiled 10 minutes before adding PCR in real time, to improve lysis, and, from spore, extract DNA with 3 kinds of different schemes (deriving from the DNA Isolator of Genosys Biotechnologies Inc., the DNAzol that derives from Helena Biosciences and Qiagen DNeasyplant mini test kit).As template, test every kind of method in order to the sporocyst that in redistilled water, diluted in 1: 10,1: 100 and 1: 1000 or DNA successively.With C-sp-f-2 and G-sp-f-2ARMS primer and contrast primer, and all use reverse Scorpion general primer, to every kind of diluent with three parts of repeated tests.Every kind of template of 5 μ l is added during PCR measures, and condition but adds 0.25 μ g/ μ lBSA (when described BSA concentration is not variable) basically as previously mentioned.With or the DNA that extracts of DNAzol or Qiagen DNA preps obtain good consistent result.

The grape that estimation is collected from field test is given birth to G the mould strain isolated of single shaft (being appointed as 17A) ₁₄₃And A ₁₄₃Gene frequency.To collect in the redistilled water from the sporocyst of 120 blades, and by centrifugal results.This sample contains have an appointment 2400 scabs and 6.4 * 10 ⁷Individual sporocyst.With 12 * 10 ⁶Individual sporocyst is kept under-80 ℃, to be ready to use in DNA extraction.Carry out extracting genome DNA with DNeasyPlant Minikit (Qiagen catalog number 69103), the DNA of gained was diluted in redistilled water with 1: 10 and 1: 100, with the template of analyzing as PCR in real time.The PCR condition as mentioned above.The cycle threshold that is produced (Ct) is 20 for the G Auele Specific Primer, is 34 for the C Auele Specific Primer, produce 14 round-robin Ct differences, so the frequency of resistance allele is about 1/10,000.

Also assessed multipleization (multiplex) method.In this case, described Scorpion measuring element is incorporated on the ARMS primer, so that can in same PCR, carry out multiple G ₁₄₃And A ₁₄₃The allelotrope pcr amplification.The Scorpion/ARMS primer sequence is as follows: G ₁₄₃Allele-specific primers: 5 ' FAMCCCGCCCTGGGATAGCCGAGAATAAATGGGCGGG (SEQ ID NO 1) MR-HEG CCTTGGTGACAAATGAGTTTTTGGAG (3 ' SEQ ID NO 118) A ₁₄₃Allele-specific primers: the described Scorpion measuring element details of 5 ' TETCCCGCCCTGGGATAGCCGAGAATAAATGGGCGGG (SEQ ID NO 119) MR-HEG CCTTGGTGACAAATGAGTTTTTGAGC3 ' (SEQ ID NO 120) as mentioned above.A ₁₄₃Allele-specific primers TET mark is distinguished between described two kinds of amplicons with permission.Use a kind of shared oppositely (unlabelled) primer: the long 95bp of amplicon that reverse 5 ' CATAACCAGTCAACAACTTCTTTTCC3 ' (SEQ ID NO 121) produces in both cases.Use Oligo5 and MFold programdesign Scorpion primer (as mentioned above) once more.Other PCR in real time component is primer concentration change during the checking of this multipleization method as mentioned above.Final concentration by reducing G FAM Scorpion is to 100nM, and the final concentration of C TET Scorpion and reverse primer is maintained 500nM, might detect the ratio (referring to Fig. 2 b) of at least 1: 500 C: G reliably with plasmid DNA as template.

Embodiment 2

In embodiment 2, we reported part standing grain powdery mildew wheat and barley cause a disease mutation cytochrome b gene sequence evaluation, cause standing grain powdery mildew wheat and barley to cause a disease in the mutation and be used for monitoring standing grain powdery mildew wheat and the barley description that the PCR in real time Scorpion of this SNP of mutation measures of causing a disease the evaluation of the SNP of the compound resistance in strobilurins analogue or the same crossed resistance group.

The strain isolated of pathogenic mutation of standing grain powdery mildew wheat and the pathogenic mutation (pathogenic agent of wheat and barley powdery mildew) of barley is collected in France the north, Germany, Ireland and Britain.This collection can be finished by one of two kinds of methods: artificially collect the field blade or (Burkard Manufacturing Co.Ltd., Rickmansworth UK) artificially collect air spore sample by fixing injection spore collector onboard.

The wheat leaf blade that infects the sporulation white powder is collected in the place that has been exposed to the strobilurins analogue from wheat population a plurality of tests.When sending the laboratory back to, blade is put into polyethylene bag and spend the night in 21 ℃ of cultivations.Spore formed sorus (pustule) again in second day.By fresh leaf section (the wheat Rapier Cultivar (cv Rapier) that places on the 9cm culture dish filter paper (Whatman No.1) that contains 1.2% water agar (water agar), 9 ages in days) rap infected blade on, with the cultivation of going down to posterity of all soruss.The flat board of inoculation was newly cultivated 7 days, tested the susceptibility of gained bacterium colony then the strobilurins analogue.

Collect about spore, downcut wheat leaf blade, place on the 1.8% water agar of plastics plate, maintain 5 ℃ and descend stand-by from 9 age in days plant (Rapier Cultivar).To spray the spore collector and be installed on the automobile top, automobile will be travelled along projected route in each country with the speed up to 90km/hr.Contain leaf section plastics plate and place spore to collect base for post portion, be deposited on the blade with the airborne spore that allows to enter collector.Every approximately 80km changes plate.In case after a collection of leaf section has been used for the spore collector, immediately it is transferred to the square culture dish (10cm that contains 60ml 1.8% water agar and filter paper ²) in, and be stored in 5 ℃.

When returning Jealott ' s Hill, the blade that will be exposed to the spore collector is cultivated in thermostatic chamber (day is long 16 hours, illumination 4-5,000lux, 21 ℃ of temperature, relative humidity on every side).In the spore collector, expose the back in the time of 5-6 days, can observe and form standing grain powdery mildew sorus (blade material a plurality of sub-districts yellow occurs aleurioconidium then and forms spot).These soruss or the leaf section in the 9cm culture dish are uploaded foster " colony " (colony of each sample phase) that be that be commissioned to train, perhaps downcut as single sorus isolate, the 15ml in the 5cm culture dish is coated with on the 1.2% water agar of filter paper and cultivates respectively respectively then.The leaf section of inoculation standing grain powdery mildew colony was cultivated 7 days, and sporulation after this is enough to be used in inoculating phenotype resistance analyte.Single spore separation thing was cultivated 7 days, cultivated once, be used for test so that enough materials to be provided but again it is gone down to posterity.If sporulation is poor, then repeat above-mentioned steps, until on all leaf sections, obtaining good (60-70%) sporulation disease coverage, be used for measuring to produce enough conidiums.

Between inoculation, handle with the aqueous solution of 5ppm (spray rate of known generation 100% control strobilurins susceptibility baseline separation strain) strobilurins analogue and Tween 20 wetting agents on 24 hours the wheat seedlings blade of taking, carry out the test of resistance isolate and keeping subsequently.With isolate or as large group (mass population) or as or single sorus isolate test.Conidia stems is inoculated on the treated leaf section.Infected material was cultivated 7 days in controlled environment (as mentioned above), estimated then.

All be considered to have potential resistance with any grower on the leaf section of 5ppm strobilurins analogue processing.The blade of further handling at the strobilurins analogue from the material of these scabs is uploaded and is commissioned to train fosterly, confirming the resistance in plant, and analyzes with the molecular assay of describing among the application.Can detect about 1/100 or higher phenotype fastness frequency by large group screening, the flat board that produces 100% disease control (responsive type) by single spore separation thing that grower and contrast (resistance) on the treated blade is suitable and treated blade wherein compares, and estimates more accurate frequency.

Use primer (Cytb3F (5 ' CAGCTTCAGCTTTCTTCT3 ') (SEQ ID NO 122) and (Cytb9R (5 ' ACTTAAAGGTCTAAATTG3 ') (SEQ ID NO 122) based on aspergillus niger (Aspergillus niger) and coarse arteries and veins spore mould (Neurospora crassa) cytochrome b gene conserved regions, described primer has been described the profile of coding based on the sequence of fungal cell's pigment b amino acid district 86-322 of yeast saccharomyces cerevisiae numbering system), the pathogenic mutation cytochrome b gene sequence of amplification part standing grain powdery mildew wheat.By directly rapping, will collect in the 1.5ml Eppendorf pipe from about 500mg conidium of the strobilurins analogue responsive type isolate that is not exposed to the selection of strobilurins analogue as yet from blade with sporulation disease.From this conidium sample, extract DNA with phenol/chloroform extraction scheme (referring to above).By existence and the quality of gel electrophoresis analysis DNA, in redistilled water, prepare serial dilution (1: 10,1: 100 and 1: 1000), as the mould material among the PCR.According to the manufacturer's of Taq polysaccharase (Gibco) suggestion, set up PCR, the end reaction volume is 100 μ l, then primer is added in the reaction, to final concentration be 1pmole/ μ l.Every kind of DNA diluent 10 μ l are added among the suitable PCR.Carry out strict program, with the danger of restriction PCR pollution.Carry out 30 circulations on Hybaid Omn-E instrument, each circulation is 94 ℃ 45 seconds, 42 ℃ 45 seconds and 72 ℃ 1 minute 30 seconds.Also carry out 94 ℃ of initial insulations of 3 minutes and 72 ℃ of last extensions of 10 minutes.By gel electrophoresis analysis 18 μ l reaction mixtures, assess the efficient of PCR then.The sample of the about 500bp PCR of 2 μ l product is cloned in TA PCR clone's pCR2.1 carrier (Invitrogen), and is transformed into Bacillus coli cells (TOP 10 One Shot according to manufacturer's suggestion ^TMCompetent cell) in.By carrying out Wizard minipreps (according to the explanation of Promega) and analyzing the restriction digest, check whether a series of institutes DCRP exists the insertion fragment with EcoRI.Insert 6 cloning and sequencings (ABI377XL automatic sequencer) of clip size (about 500bp) with M13 forward primer and reverse primer to having expection then.When the nucleotides sequence column data analyzed with the associated biomolecule information software from these researchs, find a kind of new cytochrome b gene that has close homology with other known ascomycetes cytochrome b sequence of new sequence encoding of gained.In table 3, can find coding G ₁₄₃The tract of 61 Nucleotide of 30 bases in the second bit base upstream and 30 bases in downstream in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).

To be used for the amplification in cytochrome b purpose zone subsequently based on the standing grain powdery mildew Auele Specific Primer of described new sequences Design.These primers are ERY11F (5 ' ATGAACAATTGGTACAGTAAT3 ') (SEQ ID NO 124) and ERY12R (5 ' GTTAGGTATAGATCTTAATAT3 ') (SEQ ID NO 125).They combine and have described profile according to the encoding sequence of the amino acid district 114-287 of fungal cell's pigment b of yeast saccharomyces cerevisiae coding scheme.

With ERY11F and ERY12R primer from standing grain powdery mildew wheat causes a disease mutation strobilurins analogue resistance colony, amplification part cytochrome b sequence.Conidium (about 200mg) is suspended in the 200 μ l redistilled waters, and in redistilled water, diluted with 1: 10,1: 100 and 1: 1000.Every kind of conidium diluent of 10 μ l is added Ready.To.Go ^TMIn the Taq polysaccharase PCR microballon (Amersham Pharmacia Biotech production number 27-9555-01), make 25 μ l, make that the primer final concentration is 1pmole/ μ l with ERY11F and ERY12R primer solution.Carry out standard program, with the danger of restriction PCR pollution.Carry out 30 circulations on Hybaid Omn-E instrument, each circulation is 94 ℃ 45 seconds, 52 ℃ 45 seconds and 72 ℃ 1 minute 30 seconds.Also comprise 94 ℃ of initial step of 3 minutes and 72 ℃ of last extensions of 10 minutes.All PCR all repeat with three parts in this case.After passing through the described PCR of gel electrophoresis analysis 10 μ l on the 0.8%TBE sepharose, before the clone, merge about 500bp PCR product of gained.Then the PCR product sample of the described merging of 2 μ l is cloned in the TA Invitrogen PCR clone pCR2.1 carrier, and is transformed into Bacillus coli cells (TOP10 One Shot according to manufacturer's suggestion ^TMCompetent cell) in.By carrying out Wizardminipreps (according to the explanation of Promega) and analyzing the restriction digest, check a series of clones' insertion fragment with EcoRI.Insert segmental 10 cloning and sequencings (ABI377XL automatic sequencer) with M13 forward primer and reverse primer to having expection size (about 500bp) then.Analysis with suitable bioinformation software generation to described sequence data, when comparing, disclose a G--＞C point mutation in described cytochrome b gene sequence in all 10 strain isolateds with the pathogenic mutation cytochrome b gene sequence of the wild-type standing grain powdery mildew wheat of previous acquisition.This DNA point mutation causes going up glycine the 143rd (according to yeast saccharomyces cerevisiae coded number system) and becomes L-Ala.

Also from causing a disease the mutation isolate, two kinds of standing grain powdery mildew barleys identify part cytochrome b gene order.Upward conidium small sample (about 100mg) is rapped the aseptic Eppendorf pipe from infected barley leaves (by before being prepared) about the described method of wheat.These conidium samples are remained on-80 ℃ to be descended stand-by.Every kind of sample is resuspended in the 200 μ l redistilled waters then, and with 1: 10 and further dilution in 1: 100, with the template of every kind of diluent of 10 μ l as amplification.With ERY11F and ERY12R primer amplification part cytochrome b gene order.PCR condition and component are as previously mentioned.When gel electrophoresis analysis, find the PCR product of a kind of expection size (about 500bp).This product cloning in TA pCR2.1 carrier, and is checked order as previously mentioned.When sequential analysis, find to remove G ₁₄₃Outside the 1bp (T becomes A) of the codon second bit base downstream 379bp, identical with the described sequence that from standing grain powdery mildew wheat causes a disease mutation, increases from the pathogenic mutation expanded cells pigment b sequence of standing grain powdery mildew barley.This sequence change will not cause the amino acid change (being silent mutation) in the final protein.

With above-mentioned project study 31 kinds of different standing grain powdery mildew isolates, in all cases, find described sequence and cause the cause a disease G of anti-strobilurins analogue resistance in the mutation of standing grain powdery mildew wheat ₁₄₃The A sudden change is consistent.In causing a disease the mutation sample, standing grain powdery mildew barley do not detect A ₁₄₃Resistance allele.

According to about G ₁₄₃The above-mentioned information of A point mutation, designed specificity ARMS standing grain powdery mildew primer: based on the forward ARMS primer of wild-type sequence: G-sp-1:CCATACGGGCAGATGAGCCACTGGAG (SEQ ID NO 126) based on G ₁₄₃The forward ARMS primer of A sudden change: C-sp-1:CCATACGGGCAGATGAGCCACTGGAC (SEQ ID NO 127) and design are positioned at the contrast primer of described point mutation upstream: STAND2:GCCATACGGGCAGATGAGCCACTG (SEQ ID NO 128)

Under the situation of G-sp-1 and C-sp-1 primer ,-1 base (3 ' end base) is all corresponding to G ₁₄₃/ A ₁₄₃Second Nucleotide of codon.The base that is different from wild-type cell pigment b standing grain powdery mildew sequence in the described primer is represented with runic.-2 become the A base from the G base.Carrying out this change is for described primer is gone to stablize, as common in the ARMS reaction.

Scorpion ^TMThe product detection system is in this case as testing mechanism.Use Oligo5 and MFold program (referring to the details among the embodiment 1) design Scorpion primer once more.The sequence of standing grain powdery mildew Scorpion primer is: 5 ' FAM- CCCGCGTTTTAGCTGCTTTAGCTTTAATGC GGCGGGThe zone of (SEQ ID NO 129) MR-HEG-AACACCTAAAGGATTACCAGATCCTGCAC3 ' (SEQ ID NO 130) underscore is that hair clip forms part, FAM is a fluorescein(e) dye, MR is the non-fluorescent quenching agent that is connected in the uridylic residue, and HEG is the hexaethylene glycol monomer that blocking-up is duplicated.The sequence that italic is represented is the reverse primer sequence, and the sequence that runic is represented is the Scorpion sequence in the extension products of described reverse primer of annealing.

All primers are synthetic by Oswel DNA service.Before using, described primer is diluted to 5 μ M respectively in 500 μ l cumulative volumes.In PCR, primer further is diluted to the final concentration of 500nM then.

At first pass through to use plasmid DNA and total DNA as template, the checking primer can be used in the ARMS/Scorpion analysis.Carry out this checking and be specificity and sensitivity in order to check described design of primers.To comprise the dna fragmentation of part wild-type cell pigment b gene order and contain G ₁₄₃The corresponding sequence of A sudden change is cloned in the TApCR2.1 carrier, to be used for this verification method.Plasmid DNA is always diluted in 1mg/ml BSA, the template in measuring as PCR in real time then.Use the phenol/chloroform extraction method, from standing grain powdery mildew wheat causes a disease mutation strobilurins analogue responsive type contrast isolate, extract the fungal DNA (as previously mentioned) that is used to analyze.Then, primer with empirical tests, under two conidium extent of dilution, test is from the pathogenic mutation conidium sample of the standing grain powdery mildew wheat of French strobilurins analogue responsive type strain isolated (F12C) with from the pathogenic mutation conidium sample of the standing grain powdery mildew wheat of German strobilurins analogue resistance strain isolated (11-8).All three kinds of fungi strain isolateds all derive from single sorus.

In all cases, the AmpliTaq Gold enzyme (Perkin-Elmer/ABI) that in reaction mixture, all comprises 1 unit/25 μ l reactants.Reaction mixture also contains 1 * damping fluid (10mM Tris-HCl (pH 8.3), 50mM KCl, 3.5mM MgCl ₂, 0.01% gelatin), 100 μ M dNTP.Amplification is carried out in ABI Prism 7700 instruments, to carry out the successive fluorescence monitoring.After 95 ℃ of initial insulations of 10 minutes, carry out 50 circulations: 95 ℃ of 15 seconds and 60 ℃ 45 seconds.At all round-robin annealing/extension stage monitoring fluorescence.

When at contrast template (plasmid and from the DNA of strobilurins analogue responsive type contrast strain isolated) test, standing grain powdery mildew ARMS/Scorpion primer shows good specificity, and no evidence shows and makes a mistake initial from the template of mistake.In Fig. 3 a, shown that mixture (second chain+standing grain powdery mildew Scorpio, G-sp-1+ standing grain powdery mildew Scorpio and C-sp-1+ standing grain powdery mildew Scorpio) with described three kinds of primers is to the amplification of 1: 100 dilution strobilurins analogue responsive type contrast standing grain powdery mildew DNA.Every kind of reaction is all carried out with double.The hyperfluorescence signal is sent in reaction of contrast primer and the reaction of G Auele Specific Primer, and the reaction of C Auele Specific Primer does not demonstrate the increase of any fluorescence.Contrast ARMS primer and G specificity ARMS primer are identified, and are incorporated into described template, and the C Auele Specific Primer does not combine with the template of existence in the reaction.In this case, G ₁₄₃: A ₁₄₃Only there is wild-type allele in the allelotrope analysis revealed.

Fig. 3 b has described the PCR that the mixture (second chain+standing grain powdery mildew Scorpion, G-sp-1+ standing grain powdery mildew Scorpion and C-sp-1+ standing grain powdery mildew Scorpion) of wherein using described three kinds of primers is analyzed French responsive type strain isolated (F12C).In each case, all will about 200mg sectional growing spore suspension in 200 μ l redistilled waters, and in redistilled water with 1: 100 and dilution in 1: 1000.The described diluent of 5 μ l is joined among the suitable PCR.When analyzing once more, strong signal is sent in reaction of contrast primer and the reaction of G Auele Specific Primer, and the reaction of C Auele Specific Primer does not demonstrate the increase of any fluorescence.This shows only detect wild-type allele in this sample.Compare as template with using plasmid DNA, when using conidium, produce the fluorescence of determining and postpone as reaction template.Or this is because can reducing as the molecule of template copy of existing in described reaction, perhaps owing to there is the inhibition component in the conidium sample.

Fig. 4 has described the PCR of two kinds of dilution German resistance strain isolateds of mixture (second chain+standing grain powdery mildew Scorpio, G-sp-1+ standing grain powdery mildew Scorpio and C-sp-1+ standing grain powdery mildew Scorpio) amplification conidium of wherein using described three kinds of primers.In this case, strong signal is sent in reaction of contrast primer and the reaction of C Auele Specific Primer, and the reaction of G Auele Specific Primer does not show any fluorescence.This shows only detect mutant G in this sample ₁₄₃A allelotrope.

Embodiment 3

In embodiment 3, we have reported the evaluation and the PCR in real time research of part rye beak spore cytochrome b gene sequence, wherein to various rye beak spore isolate screening G ₁₄₃The A resistance allele.This is to carry out described G on the bacterial classification that does not wherein formerly identify described point mutation as yet ₁₄₃The embodiment that A measures.

Wild-type rye beak spore strain isolated is collected in Britain and France (referring to table 8: rye beak spore strain isolated detail file).K1124 and K3327 strain isolated can be considered to " baseline " (collecting) before the strobilurins analogue is used in the field, and other strain isolated gets and has been exposed to the test site of for several times spraying the strobilurins analogue in comfortable a plurality of season.Manually choose infected barley leaves, be stored in the polyethylene bag and deliver to Jealott ' s Hill Research Station (Zeneca Agrochemicals).When arriving the laboratory, downcut single scab from blade, in ethanol, carry out surface sterilization (30 seconds), then washing (2 minutes) in 0.1% chlorine bleach liquor, place then on the Lima Bean agar (referring to table 9), (UK)/12 hour no photoperiod, 19 ℃ of constant temperature were cultivated 4-5 days down for 365nmPhilips TLD 18W/08-Philips Lighting Ltd, Croydon replacing 12 hours black light.The bacterium colony that grows on scab is as the spore suspension of no count, uploads at Lima Bean agar and is commissioned to train fosterly, and cultivates as mentioned above about 7 days, until reaching sporulation.Take out the gained spore, it is stored in the liquid nitrogen, until the needs strain isolated.By with the spore suspension plating to Lima Bean agar, and cultivate about 7 days as mentioned above until reaching sporulation, reach the recovery of strain isolated.

The strain isolated code	Source country
		K1124	Britain
K3327	Britain
		K3274	Britain
K3276	Britain
		K3278	Britain

Table 8: the detail file of rye beak spore strain isolated

From two rye beak spore strain isolateds (K1124 and K3327), obtain part cytochrome b gene order.These strain isolateds are by 100 in the substratum that is inoculated into no fermentable carbon source, 000 spore/ml suspension grows out with 19 ℃, 85rpm shaking culture 21 days (12 hours illumination/12 hour dark), filter the collection mycelium through Miracloth, and freezing stand-by in-20 ℃.By with phenol/chloroform extraction scheme (referring to embodiment 1), prepare DNA from mycelium.Check DNA output and quality by gel electrophoresis, prepare continuous storing solution diluent (in redistilled water 1: 10,1: 100 and 1: 1000) then as the template in the pcr amplification.Described in previous embodiment, set up PCR.Used primer or according to degenerated primer (deg4F (5 ' AGGTYTRTAYTRYGGDTCWTA3 ') (SEQ ID NO 131) and the deg3R (5 ' AGCDATAACWCCTAATAATTT3 ') (SEQ ID NO 132), or standing grain powdery mildew Auele Specific Primer ERY11F and ERY12R (as detailed description among the embodiment 2) of the homologous region of fungal cell's pigment b gene (having described the sequence of coding) design according to the amino acid district 100-294 of yeast saccharomyces cerevisiae coding scheme.Use two kinds of primers right then,, every kind of PCR product cloning is arrived in the pCR2.1 TA carrier (Invitrogen) from the band of each strain isolated expection size of amplification (about 500bp).Carry out Wizard minipreps and have expection size (about 500bp) with evaluation and insert segmental clone, and submit 5 clones to, check order from each clone's incident with use M13 forward primer and reverse primer.When analyzing, find to have identified a kind of and the closely-related new cytochrome b gene sequence of other ascomycetes cytochrome b gene sequence with the associated biomolecule information software.In table 3, can find coding G ₁₄₃The tract of 61 Nucleotide of 30 bases in the second bit base upstream and 30 bases in downstream in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).

At G ₁₄₃Designed different specificity ARMS rye beak spore primers around the A point mutation site: based on two kinds of forward ARMS primer: G-sp-2:CCTTATGGACAGATGTCTTTATGATG (SEQ ID NO 133) G-sp-3:CCTTATGGACAGATGTCTTTATGAAG (SEQ ID NO 134) of wild-type sequence G based on expection ₁₄₃Two kinds of forward ARMS primer: C-sp-2:CCTTATGGACAGATGTCTTTATGATC (SEQ ID NO 135) C-sp-3:CCTTATGGACAGATGTCTTTATGAAC (SEQ ID NO 136) of A sudden change and design are arranged in the contrast primer of described point mutation upstream: STAND3:TCCTTATGGACAGATGTCTTTATG (SEQ ID NO 137) at described ARMS primer, and-1 base (3 ' end base) is corresponding to G ₁₄₃/ A ₁₄₃Second Nucleotide of codon.The base that is different from rye beak spore wild-type cell pigment b sequence in the described primer is represented with runic.-2 become A base or T base from the G base.Carrying out this change is for described primer is gone to stablize, common as the ARMS primer.

Scorpion ^TMThe product detection system as testing mechanism, is used Oligo5 and MFold programdesign Scorpion reverse primer (referring to the details among the embodiment 1) in this case once more.The sequence of rye beak spore Scorpion primer is: 5 ' FAM-CCCGCCATATTAGCTGCATTAGTATTAATGCGCCGGG (SEQ ID NO 138)-MR-HEG-TACACCTAAAGGATTACCTGACCCTGCAC3 ' (SEQ ID NO 139) (relevant details is referring to previous embodiment).

All primers are synthetic by Oswel DNA Service.Before using, described primer is diluted to 5 μ M respectively in 500 μ l cumulative volumes.In PCR, primer further is diluted to the final concentration of 500nM then.

The plasmid DNA of at first passing through to use different concns is as template, and the checking primer can be used in the ARMS/Scorpion analysis.Carry out this checking and be specificity and sensitivity in order to check described design of primers.With part wild-type cell pigment b gene order with contain G ₁₄₃The corresponding sequence of A sudden change is cloned in the TApCR2.1 carrier, to be used for this verification method.Owing in rye beak spore DNA, do not find this sudden change as yet, therefore use the site-directed mutagenesis strategy with A ₁₄₃Point mutation is incorporated in the described sequence, is about to described point mutation and is incorporated in a kind of design of primers, clones as template, with this primer amplification purpose district with wild-type then.Set up PCR with the aforesaid standards method, carry out 30 circulations on Hybaid Omn-E instrument, each circulation is 94 ℃ 45 seconds, 56 ℃ 45 seconds and 72 ℃ 1 minute 30 seconds.Also comprise 94 ℃ of initial insulations of 3 minutes and one 72 ℃ 10 minutes last extension insulations.With the PCR product cloning of about 370bp in the TApCR2.1 carrier, and before being used for this experiment, to the gained cloning and sequencing, to check any PCR inductive mistake.

The concentration that calculates undiluted described plasmid DNA prepared product is about 2 * 10 ¹¹Molecule/μ l.Therefore two kinds of plasmid storing solutions are diluted to 2 * 10 in 1mg/ml BSA ⁷, 10 ⁵, 10 ³With 10 ¹Molecule/μ l uses every kind of diluent of 5 μ l equal portions then, produces about 1 * 10 in corresponding PCR ⁸, 10 ⁶, 10 ⁴With 10 ²The molecule plasmid.PCR condition and component are as previously mentioned.In Fig. 5 a, demonstrate the serial dilution degree that detects the G plasmid with the G primer mixture, with every kind of 10 times of plasmid diluents, fluoroscopic examination postpones about 4 circulations.Fig. 5 b shows G (wild-type (Wt)) and C (mutant) box with the maximum concentration of G-sp-2 primer mixture amplification.Even described dna profiling concentration height is (in the reaction about 10 ⁸Molecular template), the G primer also is period very late in PCR, just leaves the C template and wrong start.Therefore remarkable delay before G wrong start incident has proved that described primer has good specificity window (window of specificity).By specificity and non-specific plasmid diluent, the C-sp-2 primer sets also demonstrates good specificity (Fig. 6 a and 6b).In following experiment, use G-sp-2 and C-sp-2 primer mixture to replace G-sp-3 and C-sp-3 primer mixture.

The second section of this research is to compare as the template of PCR with total (genome) DNA and cDNA.From rye beak spore strain isolated, prepare total DNA material with phenol-chloroform extraction method (as previously mentioned).With RNeasy Plant minikit (Qiagen catalog number 74903),, extract total RNA from 100mg ground mycelium according to manufacturer's suggestion.Use Advantage RT-PCR test kit (Clontech catalog number K1402-1) then,,, carry out the synthetic of the first chain cDNA with RT PCR with the total RNA of 1 μ g according to manufacturer's suggestion.Preparation is from the total DNA and the cDNA storehouse of three strain isolateds (K3278, K3274 and K3276).Total dna library as template, with 1: 100,1: 1000 and dilution in 1: 10000, and is used as pure template with cDNA, with 1: 5 and dilution in 1: 10.In each case, 5 μ l templates are added among the PCR.Also use the condition of the PCR in real time of describing in embodiment 1 and 2, just carry out 40 round-robin PCR at this moment, rather than 50 circulations.Fig. 7 a, 7b and 7c have illustrated with three extent of dilution (extent of dilution 1: total DNA (1: 100) and cDNA (pure); Extent of dilution 2: total DNA (1: 1000) and cDNA (1: 5); Extent of dilution 3: total DNA (1: 10000) and cDNA (1: 10)) total DNA and cDNA template, the result who obtains with the amplification of G primer mixture.Fig. 8 a, 8b and 8c have described total DNA and the cDNA template with the amplification of C primer mixture.Use total DNA and without cDNA as template, obtain sensitive result more and cycle threshold more early.For the chance that offers the best detects any C sudden change, selecting extent of dilution is that total DNA input thing of 1: 10 and 1: 1000 is used for analysis in the future.When C Auele Specific Primer mixture being used for this 40 round-robin PCR, can not detect change in fluorescence, show and in these strain isolateds, only find wild-type G ₁₄₃Allelotrope.

Embodiment 4

In embodiment 4, we have reported and have partly justified the evaluation of nuclear cavity bacteria cytochrome b gene sequence and wherein multiple round nuclear cavity bacteria strain isolated is carried out G ₁₄₃The research of A resistance allele detection assay.This is wherein before not identified G as yet ₁₄₃The bacterial classification of A sudden change.This embodiment is divided into two parts: a part is described a kind of PCR in real time research of using the cDNA prepared product of a kind of intercalative dye detection method and circle nuclear cavity bacteria strain isolated; And another part is described in the PCR in real time research of using the Scorpion detection assay on the genomic dna prepared product of round nuclear cavity bacteria strain isolated.Use the PCR in real time research of intercalative dye:

Circle nuclear cavity bacteria (pathogenic agent of barley filigree) wild-type strain isolated during 1994,1996 and 1998, be collected in Britain and France (referring in the table 10 about the detailed description of circle nuclear cavity bacteria strain isolated).The strain isolated of 1994 and 1996 can be considered to " baseline " (collecting) before the strobilurins analogue is used in the field, and strain isolated in 1998 gets and has been exposed to the test site of spraying the strobilurins analogue for several times in comfortable a plurality of season.Manually choose infected barley leaves, and deliver to Jealott ' s Hill Research Station (ZenecaAgrochemicals).When arriving the laboratory, blade was cultivated 24-48 hour in 21 ℃ in wet environment.Downcut single scab from blade, in ethanol, carry out surface sterilization (30 seconds), then washing (2 minutes) in 0.1% chlorine bleach liquor places on the Rose Bengal agar (referring to table 9) then, is alternately cultivating 4-5 days under 12 hours black light/12 hour unglazed systems, 22 ℃ of constant temperature.All substratum of describing in the table 9 all by sterilizing with the 15psi autoclaving under 121 ℃ in 15 minutes.The bacterium colony that grows on scab is uploaded at V8+ Streptomycin sulphate agar (referring to table 9) by the mycelium plug and is commissioned to train fosterly, and cultivates as mentioned above 14 days.

Substratum:	Composition:
			Potato glucose meat soup:	Potato glucose (Difco) deionized water	??24g ??1000ml
Lima Bean agar:	No. 3 agar of Lima Bean agar (Difco) (Oxoid) deionized water	??23g ??10g ??1000ml
			Rose Bengal agar:	Add behind No. 3 agar of glucose (Oxoid) yeast extract paste (Oxoid) (Oxoid) the deionized water autoclaving; Vetstrep (Sigma) Rose Beagal (Sigma)	??10g ??2g ??17g ??1000ml ??125mg ??25mg
V8+ Streptomycin sulphate agar	V8 juice (Campbells) lime carbonate (Fisher)	??200ml ??3g

	Add behind No. 3 agar (Oxoid) the deionized water autoclaving; Vetstrep (Sigma)	????20g ????800ml ????200mg
			Czapek Dox V8 agar	No. 3 agar of V8 juice (Campbells) lime carbonate (Fisher) Czapek Dox agar (Oxoid) (Oxoid) deionized water	????200ml ????3g ????45.5g ????7g ????800ml
Supplier:	Campbells?Groceries，Kings?Lynn，Norfolk， UK Oxoid，Basingstoke，UK Sigma?Chemicals，Poole，UK Difco，Detroit，Michigan，USA Fisher?Scientific，Loughborough，UK

Table 9: culture medium prescription

Take out the mycelium material and the spore material that are produced, be stored in the liquid nitrogen until the needs strain isolated.Be inoculated on the V8 agar by the fungi sample plate that will store, and cultivate 14 days as mentioned above, reach the recovery of strain isolated until reaching sporulation.

The strain isolated code	The age of collecting	The country in source
			K1056	?1980	Ireland
K1916	?1994	Britain
			K2346	?1996	France
K2383	?1996	Britain
			K2385	?1996	Britain
K2390	?1996	Britain
			K2396	?1996	Britain
K3230	?1998	Britain
			K3237	?1998	Britain
K3238	?1998	Britain
			K3253	?1998	Britain

Table 10: the detail file of circle nuclear cavity bacteria strain isolated

From two strain isolateds (K1056 and K1916), identify part cytochrome b sequence.Described original wild-type sequence derives from the biological sample for preparing by the mycelium suspended liquid inoculation potato glucose meat soup (referring to table 9) with dipping.Flask under 12 hours white light/12 hour unglazed systems, was cultivated 21 days with 85rpm, 19 ℃ of constant temperature on orbital shaker, until there being enough mycelium materials to be used for the DNA extraction scheme.Filter by Miracloth then and collect mycelium, and it is freezing stand-by in-20 ℃.(referring to embodiment 1) prepares total DNA with the phenol/chloroform extraction scheme.After checking DNA output and quality by gel electrophoresis, preparation serial dilution (in redistilled water 1: 10,1: 100 and 1: 1000) is as pcr template.Test many different primers (Auele Specific Primer and degenerated primer) combination, in most of the cases do not obtain amplified production.Yet, (Cytb3F and Cyt9R, relevant details is referring to embodiment 2) produced a kind of about 500bp PCR product really according to a kind of primer of the mould conservative cytochrome b sequences Design of aspergillus niger and coarse arteries and veins spore.This product cloning in TA pCR2.1 carrier (Invitrogen), is checked order to 6 clones with M13 forward primer and reverse primer.Described as a result the time when analyzing, find that reverse primer correctly is incorporated into the cytochrome b sequence, and forward primer wrong start in having the dna sequence dna that includes the increment feature.Design specificity circle nuclear cavity bacteria primer in the tract of described new cytochrome b gene (Pt2R:5 ' CTT ACA TCT GTAATA GGT AAT3 ') (SEQ ID NO 140), and with its be used from round nuclear cavity bacteria cDNA template according to the forward primer of venturia inaequalis cytochrome b gene sequences Design (Pt5F:5 ' TGT TAC TTT AGC AATGCA CTA3 ') (SEQ ID NO 141).From the mycelium sample of these two kinds of strain isolateds, prepare cDNA.With RNeasy test kit (Qiagen),, from 100mg ground mycelium, extract total RNA according to manufacturer's suggestion.With AdvantageRT-PCR Clontech test kit (according to manufacturer's suggestion), carry out the first chain cDNA with RT PCR from the total RNA of 1 μ g and synthesize.Then 5 μ l gained cDNA are used for PCR.PCR component and condition are as previously mentioned.Amplify about 800bp PCR product (having described the sequence of coding) of two kinds of strain isolateds according to the amino acid district 48-310 of fungal cell's pigment b of yeast saccharomyces cerevisiae numbering system.In both cases, all with the PCR product cloning in the TA pCR2.1 carrier (Invitrogen), with M13 forward primer and reverse primer 4 cloning and sequencings that contain expection size insertion fragment (about 800bp) to each strain isolated.The sequence data analysis revealed has been isolated a kind of and the closely-related new cytochrome b gene sequence of other known ascomycetes cytochrome b sequence.In table 3, can find coding G ₁₄₃The tract of 61 Nucleotide of the cDNA sequence of 30 bases in the second bit base upstream and 30 bases in downstream in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).

At G ₁₄₃Designed specificity ARMS circle nuclear cavity bacteria primer around the A point mutation site: based on the forward ARMS primer of wild-type sequence: G-sp-4:CCCTACGGGCAAATGAGCCTTTGAAG (SEQ ID NO 142) based on possible G ₁₄₃The forward ARMS primer of A sudden change: C-sp-5:CCCTACGGGCAAATGAGCCTTTGATC (SEQ ID NO 143) and design the contrast primer that is positioned at described point mutation upstream: the used reverse primer of STAND4:ACCCTACGGGCAAATGAGCCTTTG (SEQ ID NO 144) is: UNLS4:TACACCTAAAGGATTTCCTGACCCTGCAA (SEQ ID NO 145) moreover, in described ARMS primer ,-1 base (3 ' end base) is corresponding to G ₁₄₃/ A ₁₄₃Second Nucleotide of codon.The base that is different from round nuclear cavity bacteria wild-type cell pigment b sequence in the described primer is represented with runic.-2 become A base or T base from the G base.Carrying out this change is for described primer is gone to stablize.

All primers are synthetic by Oswel DNA Service.Before using, described primer is diluted to 5 μ M respectively in 500 μ l cumulative volumes.In PCR, primer further is diluted to the final concentration of 500nM then.

In this case, detect described PCR product with a kind of intercalative dye.Used dyestuff is that (USA), this dyestuff combines with double-stranded DNA the YO-PRO-1 dyestuff for Molecular Probes, Seattle Washington, and launches the fluorescence that available ABI Prism 7700 instrument detecting arrive.

The plasmid DNA of at first passing through to use different concns is verified primer as template.Carry out this checking and be specificity and sensitivity in order to check described design of primers.With part wild-type circle nuclear cavity bacteria cytochrome b gene sequence and the G that contains by the site-directed mutagenesis introducing ₁₄₃The corresponding sequence of A sudden change is cloned in the TA pCR2.1 carrier, to be used for this verification method (as described in example 3 above).

The concentration that calculates undiluted described plasmid DNA storing solution is about 2 * 10 ¹¹Molecule/μ l.Two kinds of plasmids are diluted to 2 * 10 ⁷, 10 ⁵, 10 ³With 10 ¹Molecule/μ l uses every kind of diluent of 5 μ l then, produces about 1 * 10 in corresponding PCR ⁸, 10 ⁶, 10 ⁴With 10 ²Molecule plasmid (relevant PCR condition is referring to embodiment 1).Unique different be that the YO-PRO-1 dyestuff is added in the reaction mixture.Described signal is disturbed in the generation of primer dimer product after the 35th circulation.The sensitivity of this detection method is lower than the Scorpion detection system, because it is subjected to the influence of background noise (for example from introducing the fluorescent emission that dimer forms) bigger.Yet the conclusion that draws is, by can drawing valuable information with this method, but must be more careful when explanation results.

In following experiment, check whether above-mentioned strain isolated exists G ₁₄₃A allelotrope.Culture of isolated strain in having the substratum of no fermentable carbon source is to obtain being used for the material that ARMS/Scorpion measures.But the inoculation of primospore suspension (1ml, 100,000 spores/ml) contain in the conical flask of meat soup.Culture is cultivated on orbital shaker as previously mentioned, and the mycelium of gathering in the crops gained then is used for the cDNA preparation.From every kind of strain isolated preparation (as previously mentioned) cDNA material.Carrying out this step is the primer that is arranged in the intron/exon group structure of the complexity of round nuclear cavity bacteria cytochrome b sequence for fear of design.

Set up all PCR as previously mentioned, and carry out 50 circulations.Unique different be that the YO-PRO-1 dyestuff is added in the reaction mixture.With pure cDNA and with the cDNA of dilution in 1: 10 as template, all 5 μ l templates are added in all cases during PCR measures.

Fig. 9 a and 9b described with three kinds of primers to carry out with double to pcr amplification results with two strain isolateds in 11 kinds of strain isolateds of two kinds of extent of dilution tests.But the G that in the sample of being tested, does not have detection level ₁₄₃The allelic situation of A.Adopt the PCR in real time research of Scorpion detection system:

Also, designed diagnostic G according to the genome organization of the round nuclear cavity bacteria cytochrome b gene in fixed coding purpose amino acid district ₁₄₃A Scorpion measures.

By a series of primers of using according to the known coded sequence and the intron sequences of identifying subsequently designs, the genomic dna prepared product is carried out pcr amplification, illustrated the intron/exon group structure around the purpose base.Use multiple different combination of primers, according to manufacturer's explanation, with Taq Extender ^TMPCR Additive and Perfect Match  PCR Enhancer (all derive from Strategene, catalog number is respectively 600148 and 600129) are used for the segmental amplification of big PCR.Carrying out initial 94 ℃ of insulations on the Hybaid Omn-E PCR instrument after 3 minutes, carry out 30 circulations, each circulation is: 94 ℃ 45 seconds, 52 ℃ 45 seconds and 72 ℃ 3 minutes.Also comprise 72 ℃ of last insulations 10 minutes.When with gel electrophoresis analysis PCR product, with primer pter23F (coding region 5 ' ACA TAG TAA TAC TGCTTC AGC3 ') (SEQ ID NO 146)-pter25R (including subarea 5 ' TAC ATT TGAGGC AAA TAT TTC (SEQ ID NO 147) 3 ') is found a 2.7kb PCR product, and pter7F (coding region 5 ' CTA CGG GCA AAT GAG CCT TTG3 ') (SEQ ID NO 148)-pter6R (coding region 5 ' CTC TGG AAC TAT CGC TGCAGG3 ') (SEQ ID NO 149) is found a 7.5kb PCR product with primer.According to manufacturer's explanation, these PCR product cloning are arrived pCR-XL-TOPO carrier (Invitrogen, catalog number K4700-10), and as previously mentioned each 2 clone that clone incident is checked order.Find G ₁₄₃Second bit base of codon is positioned at 3 ' end of a 36bp exon.In table 3, can find coding G ₁₄₃The tract of 61 Nucleotide of 30 bases in the second bit base upstream and 30 bases in downstream in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).

On the forward chain, design a series of 31bpARMS primers and a kind of contrast primer (mutant and wild-type allele are used to increase), and on the reverse strand of intron sequences, designed a kind of shared Scorpion primer.Long 121bp of the amplicon of gained (using the ARMS primer) and 123bp (with the contrast primer).In this case, G ₁₄₃Second bit base leaves only 1bp of 3 ' splice site in the codon on the exon of a 36bp.Use Oligo5 and MFold program (referring to the detailed description among the embodiment 1) design Scorpion primer once more.Circle nuclear cavity bacteria Scorpion primer sequence is: the description of 5 ' FAM CCC GCC GCA AGC TGA TTT CAT AGG CGG G (SEQ ID NO 150) MR-HEG TTCAA GTA CAT CCA ATT TCA ATA TAC ACT3 ' (SEQ ID NO 151) Scorpion primer, primer is synthetic and PCR in real time condition such as previous embodiment described in.

In the optimization that this Scorpion measures, the specificity of more primer among the previous embodiment of ratio of test, every kind of primer has different base mismatch at its 3 ' end, stablizes (giving prominence to runic) to cause, and is as follows.

Primer	Sequence
		STAND6	A?CCC?TAC?GGG?CAA?ATG?AGC?CTT?TGA?G(SEQ?ID?NO?152)
PT-G-1	CCC?TAC?GGG?CAA?ATG?AGC?CTT?TGA?AG(SEQ?ID?NO?153)
		PT-C-1	CCC?TAC?GGG?CAA?ATG?AGC?CTT?TGA?AC(SEQ?ID?NO?154)
PT-G-2	CCC?TAC?GGG?CAA?ATG?AGC?CTT?TGA?CG(SEQ?ID?NO?155)
		PT-C-2	CCC?TAC?GGG?CAA?ATG?AGC?CTT?TGA?CC(SEQ?ID?NO?156)
PT-G-3	CCC?TAC?GGG?CAA?ATG?AGC?CTT?CGA?AG(SEQ?ID?NO?157)
		PT-C-3	CCC?TAC?GGG?CAA?ATG?AGC?CTT?CGA?AC(SEQ?ID?NO?158)
PT-G-4	CCC?TAC?GGG?CAA?ATG?AGC?CTT?TTA?CG(SEQ?ID?NO?159)
		PT-C-4	CCC?TAC?GGG?CAA?ATG?AGC?CTT?TTA?CC(SEQ?ID?NO?160)
PT-G-5	CCC?TAC?GGG?CAA?ATG?AGC?CTT?TGC?GG(SEQ?ID?NO?161)
		PT-C-5	CCC?TAC?GGG?CAA?ATG?AGC?CTT?TGC?GC(SEQ?ID?NO?163)

Table 11: circle nuclear cavity bacteria ARMS primer

1 * 10 ⁷Carry out initial primer checking on the wild-type of molecule/reaction and the mutant plasmid template.Every kind of plasmid template is carried out PCR in real time with double on ABI Prism 7700 instruments, use G specificity ARMS primer, C specificity ARMS primer and contrast primer (contrast primer both increased mutant allele, wild-type allele also increases) and common oppositely Scorpion primer.

Seen in table 12, when at mutant plasmid template and wild plasmid template test, different ARMS design of primers produces different specificity windows.When comparing with other primer of being tested, PT-G-1 and PT-C-5 provide the wideest specificity window.Therefore, select them right as the preferred primer of this mensuration.The Ct value of primer PT-C-5 on correct template is 16, and the Ct value of PT-G-1 on correct template also is 16.The PT-C-5 primer is at the 34th circulation time wrong start on wrong template, produce 18 round-robin specificity windows, and the PT-G-1 primer produces 16 round-robin specificity windows at the 32nd circulation time wrong start, the sensitivity that equals described two kinds of primers 10 times the difference of having an appointment.

Primer	Ct on correct template	Ct on incorrect template	Specificity window (circulation)
				????PT-G-1	????16	????32	????16
????PT-G-2	????23	????38	????15
				????PT-G-3	Failure	Failure	Failure
????PT-G-4	????32	????42	????10
				????PT-G-5	????22	????36	????14
????PT-C-1	????20	????34	????12
				????PT-C-2	????20	????34	????12
????PT-C-3	????28	????38	????10
				????PT-C-4	????34	????38	????4
????PT-C-5	????16	????34	????18
				????C-SP-5	????18	????34	????18

The checking of table 12:ARMS primer

Therein all in 1mg/mlBSA with the mutant plasmid with 1: 1,1: 10,1: 100,1: 1,000,1: 10,000 and 1: 100, in the plasmid admixture experiment of 000 frequency admixture wild plasmid background, test PT-G-1 and PT-C-5 ARMS primer.In all cases, total plasmid concentration of each frequency all was 1 * 10 during PCR measured ⁷Molecule/reaction.PCR in real time condition and component are as previously mentioned.

In following table, summed up the gained data:

Pcr template	PT-C-5 Ct value
		The 100%C template	????18
????1∶1	????19
		????1∶10	????22
????1∶100	????26
		????1∶1000	????30
????1∶10000	????34
		????1∶100000	????36
The 100%G template	????36

Table 13: admixture result of experiment

Because the C plasmid reduces in the wild plasmid background, fluoroscopic examination is delayed.With every kind of 10 times of diluents of C plasmid, in fluoroscopic examination, postpone 4 circulations.10, the PT-C-5 cycle threshold of a mutant molecules can be clear that in the background of 000 wild type molecule, but 1: 100, PT-C-5 primer wrong start and 100% wild-type (G) box can not be made a distinction for 000 time, therefore covered lower C: the G frequency.This shows, in this case the ARMS conversion will allow to detect credibly≤1: 10,000 frequency.

Real-time Scorpion PCR with new optimization measures, and has tested a series of round nuclear cavity bacteria sample (Ir 5-8, Ir 9-13, Ir 14-17, Ir 18-21, Ir30-34) that is collected in Irish Cork.The strain isolated of respectively organizing of these 4 or 5 strain isolateds is inoculated on the V8 agar plate, cultivates until reaching the sporulation of gained bacterium colony.

Mycelium is collected from agar plate in the aseptic redistilled water of 10ml.Mycelium suspended liquid is transferred in the 15ml Falcon pipe, and with 3200rpm centrifugal 10 minutes.Remove and anhydrate, mycelia clump (mass) is assigned in two aseptic Eppendorf pipes.Pass through with 13 centrifugal 5 minutes of 000rpm, precipitation mycelium then.Remove supernatant liquor with transfer pipet, one of them mycelium precipitation be used for DNA extraction, and with another mycelium precipitation be stored in-80 ℃ stand-by.With Qiagen DNeasy Plant Mini Kit scheme, carry out DNA extraction according to described in manufacturer's the scheme, with DNA isolation from plant tissue.With 10 times and 100 times of DNA dilutions,, during measuring, uses each PCR the DNA of 5 μ l equal portions to be used for described mensuration.With every kind of primer to (PT-C-5, PT-G-1 and contrast primer; Every kind of primer all uses with the common reverse primer that contains the Scorpion detection system), test all strain isolateds with three parts of repetitions and two extent of dilution.

Below shown the result who screens strain isolated with 1: 10 diluent of genomic dna as template:

Strain isolated	Cycle threshold
					????C	????G	????S
????Ir5-8(1)	????-	????20	????20
				????Ir5-8(2)	????-	????22	????22
????Ir5-8(3)	????34	????20	????20
				????Ir9-13(1)	????-	????18	????18
????Ir9-13(2)	????-	????20	????20
				????Ir9-13(3)	????36	????20	????20
????Ir14-17(1)	????36	????18	????18
				????Ir14-17(2)	????-	????22	????22
????Ir14-17(3)	????36	????18	????18
				????Ir18-21(1)	????-	????20	????20
????Ir18-21(2)	????-	????20	????20
				????Ir18-21(3)	????34	????18	????18
????Ir30-34(1)	????-	????20	????20
				????Ir30-34(2)	????34	????20	????20
????Ir30-34(3)	????-	????19	????19

Table 14: circle nuclear cavity bacteria PCR in real time result

In most of the cases, the PT-C-5 primer only demonstrates the cycle threshold (Ct) after the 34th circulation or the 34th circulation, demonstrate at this moment on plasmid template, make a mistake initial.In some cases, the PT-C-5 primer does not have wrong start at all.These results prove do not have evidence to be presented in the sample of being studied and have G ₁₄₃The A sudden change.

Embodiment 5

In embodiment 5, we have reported the evaluation of part grape snag h pigment b gene order.Grape snag shell is the pathogenic agent of uncinula necator.

During 1999, collect infected grape leave and fruit, and deliver to Jealott ' s Hill Research Station (ZenecaAgrochemicals) from France and gondola experiment place and commercial vineyard.After arriving the laboratory, mycelium and spore are transferred on the unsalted surface disinfectant blade of taking from 6-7 leaf seedling (Ohanez mutation) with pencil.Handle as an independent colony from each grape snag shell of collecting the place.

When receive in the laboratory, place 21 ℃ of thermostatic chambers to cultivate 2-3 weeks in the blade of inoculating.In case detect the sporulation disease, then inoculate the 3-4 leaf grape seedling of directly in plastic and plant breeding device, sowing with infected blade.With compressed-air line (compressed airline) conidium of each strain isolated is blown on the suitable target blade of 40 grape seedlings at the most from the source blade.To cover to place again and breed on the device, then it be cultivated for 2 weeks in the controlled environment growth room.Stand-by under being collected in the conidium that produces on these plant, and being frozen in-80 ℃.

With the RNeasy test kit that derives from Qiagen (according to manufacturer's suggestion), derive from from 100mg and to extract total RNA the ground grape snag conchospore of a strain isolated.With Advantage RT-PCR Clontech test kit (according to manufacturer's suggestion), synthesize the first chain cDNA from the total RNA of 1 μ g with RT PCR.Then with the template of 5 μ l cDNA as pcr amplification, use ERY 11F (5 ' ATGAACAATTGGTACAGTAAT3 ') (SEQID NO 163) and ERY 4R (5 ' AAATCTGTTAAAGGCATAGCC3 ') (SEQID NO 164), according to the yeast saccharomyces cerevisiae numbering system, described primer has been described the profile of the amino acid/11 14-309 of fungal cell's pigment b.Amplify a dna fragmentation of expection size (about 500bp), described PCR product cloning is arrived in the pCR2.1 TA carrier (Invitrogen) according to manufacturer's suggestion.Carry out Wizard minipreps, have the segmental clone of suitable insertion, submitted 5 clones to, so that check order with M13 forward primer and reverse primer with evaluation.When analyzing, find to have identified a kind of and the closely-related new cytochrome b gene sequence of other ascomycetes cytochrome b sequence with associated biomolecule information software (Seqman and Macaw).In table 3, can find coding G ₁₄₃The tract of 61 Nucleotide of the cDNA sequence of 30 bases in the second bit base upstream and 30 bases in downstream in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).According to described new sequences Design specificity grape snag shell primer, to be directly used in the spore material.Suspection is at purpose district (G ₁₄₃500bp around the SNP) there is intron sequences in, because the pcr amplification that before carries out is not succeeded on biomaterial.Use multiple different combination of primers, with Taq Extender ^TMPCR Additive and Perfect Match  PCREnhancer (all deriving from Strategene) are used for (according to manufacturer's explanation) big segmental amplification of PCR.Carrying out initial 94 ℃ of insulations on the Hybaid Omn-E PCR instrument after 3 minutes, carry out 30 circulations, each circulation is: 94 ℃ 45 seconds, 52 ℃ 45 seconds and 72 ℃ 3 minutes.Also comprise 72 ℃ of last insulations 10 minutes.Combination of primers 2F (5 ' GTT TTACCC TAC GGG CAG ATG3 ') (SEQ ID NO 165)-5R (5 ' AAA GAATCT GTT TAA GGT TGC 3 ') (SEQ ID NO 166), 2F6R (5 ' AAA CCACCT CAA AGA AAC TCC 3 ') (SEQ ID NO 167) and 4F (5 ' CAT GAATAG GAC AAG ATA TCG 3 ') (SEQ ID NO 168)-6R length range that successfully increased is the PCR product of 1.6kb-3kb.These PCR product cloning are arrived in the TA pCR2.1 carrier (Invitrogen), and check order as previously mentioned with clone subsequently.For implementing the primer walking strategy corresponding to one of every kind of different PCR products clone, this three clones' sequential analysis is shown, in described PCR fragment, there are two introns (length is 1.6kb and 1.1kb), G ₁₄₃Second bit base of codon is positioned at an intron splice site upstream 10bp.In table 3, can find coding G ₁₄₃The tract of 41 Nucleotide of 10 bases in the second bit base upstream and 30 bases in downstream in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).

Embodiment 6:

In embodiment 6, we have reported the evaluation of part monofilament h pigment b gene order and have caused a single nucleotide polymorphism for the compound resistance in strobilurins analogue and the same crossed resistance group.The monofilament shell is the pathogenic agent of cucurbit Powdery Mildew.

Collect cucumber and muskmelon blade that the monofilament shell infects from the field, with pencil with conidium dried being inoculated on the fresh leaf material (cucumber and muskmelon) in the laboratory.With the cultivation of going down to posterity of single conidium (monoconidial) strain isolated, and in plant, test by preventative differentiating solvent flow measurement in 24 hours (100ppm dosage, known generation 100% control wild type strain) at the most.To transfer in the suitable containers from the conidium of candidate's resistance strain isolated with the vacuum pump suction.Analyze the part of this sample with pcr analysis, rest part is tested again, to confirm the phenotype resistance.

On about 100mg conidium strobilurins analogue responsive type sample, implement RT-PCR strategy (as described in example 3 above).With the cDNA of gained as the template in the pcr amplification reaction, use primer Ery2F (5 ' TCACCTAGAACATTAACATGA3 ') (SEQID NO 169) and 4R (5 ' AAATCTGTTAAAGGCATAGCC3 ' (SEQ ID NO170)), according to the yeast saccharomyces cerevisiae numbering system, described primer has been described the profile of the amino acid/11 08-309 of fungal cell's pigment b.Other PCR component and condition are as previously mentioned.During the gel electrophoresis analysis of described PCR product, find the PCR product of a kind of expection size (about 600bp), then this product cloning is arrived in the TA pCR2.1 carrier (Invitrogen), as previously mentioned 5 clones with correct size insertion fragment (about 600bp) are checked order with M13 forward primer and reverse primer.When analyzing, find to have identified a kind of and the closely-related new cytochrome b gene sequence of other ascomycetes cytochrome b sequence with associated biomolecule information software (Seqman and Macaw).Design specificity monofilament shell PCR primer then, be used for from genomic dna amplification G ₁₄₃The district.In pcr amplification, use primer to SF1 (5 ' TTCCCTTCGGTCAAATGTCGC3 ') (SEQ ID NO 171)-SF8 (5 ' AAACCCCCTCAGAGAAACTCC3 ') (SEQ ID NO 172) and SF1-SF10 (5 ' GACCCCGCGCTATCATGTAAG3 ') (SEQ ID NO 173), use the spore sample that is resuspended in the redistilled water as template.Described in PCR component and condition such as the previous embodiment.By gel electrophoresis analysis PCR product, with the SF1/8 primer to finding a kind of about 2kb product, and with the SF1/10 primer to finding treaty 2.1kb band.These two kinds of PCR product cloning are arrived in the TA pCR2.1 cloning vector (Invitrogen), and as previously mentioned 5 clones are checked order.Adopt the primer walking strategy, two clones from these two clone's incidents are checked order entirely.When with associated biomolecule information software analytical sequence data, find at G ₁₄₃9bp place, the codon second bit base downstream, the intron of a 1917bp of existence.In table 3, can find coding G ₁₄₃The cDNA sequence of 37 nucleotide sequence sections of 6 bases in the second bit base upstream and 30 bases in downstream and genome sequence (having considered intron/exon group structure) in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).

Part cytochrome b gene order also increases from conidium strobilurins analogue resistance sample.With the conidium sample (＜50mg) be resuspended in the 200 μ l redistilled waters, and in redistilled water with 1: 10,1: 100 the dilution.With the template of every kind of diluent of 10 μ l, use SF1/SF8 specificity monofilament shell primer as pcr amplification.With Taq Extender ^TMPCR Additive and Perfect Match  PCR Enhancer (all deriving from Strategene) are used for this big PCR fragment of (according to manufacturer's explanation) amplification.Carrying out initial 94 ℃ of insulations on the Hybaid Omn-E PCR instrument after 3 minutes, carry out 30 circulations, each circulation is: 94 ℃ 45 seconds, 50 ℃ 45 seconds and 72 ℃ 5 minutes.Also comprise 72 ℃ of last insulations 10 minutes.Analyze described PCR product with gel electrophoresis analysis, find the band (about 2kb) of an expection size.It is cloned in the TA pCR2.1 carrier (Invitrogen), and described in previous embodiment, checks order having segmental 10 clones of described expection size insertion.Analyze described sequence data with suitable bioinformation software (Seqman, Editseq and Macaw software), when relatively the time, disclosing a G--＞C point mutation in the described cytochrome b sequence with wild-type sequence under the situation in 10 at all.This DNA point mutation causes the 143rd (according to yeast saccharomyces cerevisiae amino acid coding scheme) last single glycine to become L-Ala.

Embodiment 7:

In embodiment 7, we have reported the evaluation of part Mycosphaerella fijensis var.difformis cytochrome b gene sequence.M.fijiensis is the pathogenic agent of the black leaf cecospora spot of banana.

Collect infected banana blade from the field, with thecaspore from blade on the artificial medium of direct inoculation to the culture dish.List cystospore (monoascosporic) strain isolated is maintained on the artificial medium, and, prepare to be used for pcr analysis by in broth culture, carrying out shake-flask culture.Collect mycelium by Micracloth, and use through acid elution and autoclaved pestle and mortar and be ground into fine powder.100mg ground mycelium is used for extracting genome DNA and the first chain cDNA synthetic (described in previous embodiment).Genomic dna as before the pcr template, was diluted with 1: 10,1: 100 and 1: 1000 in redistilled water.With 5 μ lcDNA and every kind of genomic dna diluent of 10 μ l template, use the degenerated primer described in the embodiment 3 right as pcr amplification.Described in PCR condition and component such as the previous embodiment.When the described PCR product of gel electrophoresis analysis, when cDNA finds the band (about 500bp) of an expection size during as template, and when genomic dna is used as template a discovery bigger band (about 1.6kb).Described two kinds of PCR product cloning are arrived in the pCR2.1 TA carrier (Invitrogen), and 5 institute's DCRP with correct size insertion fragment (about 500bp and 1.6kb) are checked order with M13 forward primer and reverse primer.When analyzing described sequence data, find that described big PCR product is at G with the associated biomolecule information software ₁₄₃The second bit base downstream 78bp contains the intron of a 1064bp in the codon.Except existing this intron in described big PCR product, dna sequence dna is identical.This cytochrome b gene sequence is accredited as new, and closely related with other ascomycetes cytochrome b sequence.In table 3, can find coding G ₁₄₃The tract of 61 Nucleotide of 30 bases in the second bit base upstream and 30 bases in downstream in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).

Embodiment 8:

In embodiment 8, we have reported the evaluation of part Cuba false downy mildew cytochrome b gene sequence.The false downy mildew of Cuba is the pathogenic agent of cucurbit oidium.

Sporocyst is washed the deionized water from infected leaf material.With spray gun the concentration of gained sporangia suspension with 10,000 spores/ml is inoculated on the fresh leaf material.Disease takes place after, the spore suspension sample that is collected in the redistilled water is centrifugal in whizzer, produce precipitation, it is stored in-80 ℃ stand-by.Then the false downy mildew sporocyst of Cuba is resuspended in the 200 μ l redistilled waters, and in redistilled water, diluted with 1: 10 and 1: 100.With the template of every kind of sporocyst diluent of 10 μ l, use primer PLAS17F and PLAS15R (referring to the details among the embodiment 1) as pcr amplification.Described in PCR condition and component such as the previous embodiment.When the described PCR product of gel electrophoresis analysis, find the band (about 500bp) of an expection size.Then with this PCR product cloning in pCR2.1 TA carrier, and 5 institute's DCRP that have correct size and insert fragment (about 500bp) are checked order with M13 forward primer and reverse primer.When analyzing, find to have identified a kind of and the closely-related new cytochrome b gene sequence of other oomycetes cytochrome b sequence with the associated biomolecule information software.In table 3, can find coding G ₁₄₃The tract of 61 Nucleotide of 30 bases in the second bit base upstream and 30 bases in downstream in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).Embodiment 9:

In embodiment 9, we have reported the evaluation of part standing grain green-ball chamber mycetocyte pigment b gene order.Bacterium is the pathogenic agent of wheat leaf rot in standing grain green-ball chamber.

Collect infected wheat leaf blade from the field, and it is cultivated in moist environment, produce to promote spore.Take out cirrus from blade, it is applied on the Czapek Dox V8 agar plate (referring to table 9), and in controlled environment, cultivated 6 days in 19 ℃.With the cultivation of further going down to posterity of single bacterium colony isolate, and in suitable medium, increase by shake-flask culture.Stand-by under taking out fungal material and remaining in-80 ℃.As described in example 1 above, 200mg ground mycelium is carried out the genomic dna preparation.Check genomic dna output and quality by gel electrophoresis, and in redistilled water, diluted with 1: 10 and 1: 100.With the template of every kind of diluent of 10 μ l, use primer Cyt3F and Cyt9R (referring to the details among the embodiment 2) as pcr amplification.PCR component and condition are as described in example 2 above.By the described PCR product of gel electrophoresis analysis, find the band (about 500bp) of an expection size.This PCR product cloning in TA pCR2.1 carrier, and is inserted segmental 5 clones and checked order containing correct size as previously mentioned.When analyzing described sequence data, find a kind of part that the new cytochrome b gene sequence of close homology is arranged with other ascomycetes cytochrome b gene sequence that demonstrates of described PCR fragment coding with the associated biomolecule information software.In table 3, can find coding G ₁₄₃The tract of 61 Nucleotide of 30 bases in the second bit base upstream and 30 bases in downstream in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).

Embodiment 10:

In embodiment 10, we have reported the evaluation of the living thorn dish of part standing grain spore cytochrome b gene sequence.It is the pathogenic agent of cereal and dogstail anthrax that standing grain is given birth to thorn dish spore.

Collect infected blade material (lawn or liver moss) from the field, take out fungal material and artificial medium upload be commissioned to train foster.Fungal material increases by shake-flask culture, results, and be stored in-80 ℃ stand-by.As described in example 1 above, 200mg ground mycelium is carried out the genomic dna preparation.Estimate genomic dna output and quality by gel electrophoresis, and in redistilled water, prepared 1: 10 and 1: 100 diluent.With the template of every kind of diluent of 10 μ l, use primer deg4F and deg3R (referring to the details among the embodiment 3) as pcr amplification.PCR component and condition are as described in example 3 above.By the described PCR product of gel electrophoresis analysis, find the band (about 500bp) of an expection size.This PCR product cloning in TA pCR2.1 carrier, and is inserted segmental 5 clones and checked order containing correct size as previously mentioned.When analyzing, find to have identified a kind of and the closely-related new cytochrome b gene sequence of other ascomycetes cytochrome b sequence with the associated biomolecule information software.In table 3, can find coding G ₁₄₃The tract of 61 Nucleotide of 30 bases in the second bit base upstream and 30 bases in downstream in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).

Embodiment 11:

In embodiment 11, we have reported the evaluation of part Colletotrichum gloeosporiodes cytochrome b gene sequence.Colletotrichum gloeosporiodes is the pathogenic agent of fruit (fruit) anthrax (for example capsicum (pepper), avocado and mango).

Collect infected vegetable material (mango or red pepper) from the field, take out fungal material and artificial medium upload be commissioned to train foster.Mycelium increases with shake-flask culture, filters results by Miracloth.Mycelium is stored in-80 ℃ stand-by.Grind mycelium with aseptic pestle and mortar, and 100mg is used for genomic dna preparation and the first chain cDNA synthetic (step is as described above described in the embodiment) through acid elution.With every kind of genome diluent of 10 μ l and the pure cDNA of 5 μ l template, use primer deg4F and deg3R (referring to the primer details among the embodiment 3) as pcr amplification.PCR component and condition are as described above described in the embodiment.By the described PCR product of gel electrophoresis analysis, find the band (about 500bp) of expection size.When with genomic dna or cDNA during as template, PCR product big or small identical, this shows in the DNA district that is increased does not have intron.In TA pCR2.1 carrier, and the correct size of containing to each clone's incident described in the embodiment is inserted segmental 5 clones and is checked order as described above with described PCR product cloning.When analyzing sequencing data, find that described PCR product is all identical in all cases, and encode a kind of and other ascomycetes sequence there is the new cytochrome b gene sequence of close homology with the associated biomolecule information software.In table 3, can find coding G ₁₄₃The tract of 61 Nucleotide of 30 bases in the second bit base upstream and 30 bases in downstream in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).

Embodiment 12:

In embodiment 12, we have reported the evaluation of part tomato powder spore cytochrome b gene sequence.The tomato powder spore is the pathogenic agent of tomato Powdery Mildew.

Collect the tomato leaf of pathology from the field, by stem grafting kind in the sedimentation tower, with conidium the fresh leaf sheet material upload be commissioned to train foster.By vacuum pump suction, will get in the aseptic Eppendorf pipe by the conidium suction of the infection growth that is produced, and be stored in-80 ℃ stand-by.As discussed previously, it is synthetic to carry out the first chain cDNA on by the RNA of 100mg spore separation, and 5 these cDNA of μ l are used for pcr amplification, uses primer Ery2-4 (referring to the details among the embodiment 6).PCR component and condition are as discussed previously.When by the described PCR product of gel electrophoresis analysis, finds a kind of big or small PCR product (about 500bp) of expecting.Clone this PCR product, and as previously mentioned 5 clones that contain correct size insertion fragment (about 500bp) are checked order.When analyzing, find a kind of and closely-related new cytochrome b gene sequence of other ascomycetes cytochrome b sequence of described PCR fragment coding with the associated biomolecule information software.In table 3, can find coding G ₁₄₃The tract of 61 Nucleotide of 30 bases in the second bit base upstream and 30 bases in downstream in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).

Embodiment 13:

In embodiment 13, we have reported the evaluation of part Tartar internal thread powdery mildew cytochrome b gene sequence.Tartar's internal thread powdery mildew is the pathogenic agent of tomato Powdery Mildew.

Collect the capsicum blade of pathology from the field, conidia stems is inoculated on the fresh leaf sheet material of whole plant.By the vacuum pump suction, will be drawn in the suitable containers from the produce conidium that infects, and use pcr analysis.Collect the capsicum blade and the tomato leaf of pathology from field and the greenhouse of Jealott ' s Hill, and infected blade material is directly used in pcr analysis.

As previously mentioned, in that carry out the first chain cDNA from the isolating RNA of the vegetable material of 100mg spore or pathology synthetic.When the vegetable material of pathology is used as starting material, by from described preparation material, removing disease-free plant tissue, enrichment fungi scab.Duplicate samples such as 5 μ l cDNA are used for pcr amplification, use primer Ery11-12 (referring to the details among the embodiment 2).PCR component and condition are as discussed previously.When by the described PCR product of gel electrophoresis analysis, finds a kind of big or small PCR product (about 500bp) of expecting.Clone this PCR product, and as previously mentioned 5 clones that contain correct size insertion fragment (about 500bp) are checked order.When analyzing, find a kind of and closely-related new cytochrome b gene sequence of other ascomycetes cytochrome b sequence of described PCR fragment coding with the associated biomolecule information software.In table 3, can find coding G ₁₄₃The tract of 61 Nucleotide of 30 bases in the second bit base upstream and 30 bases in downstream in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).

Embodiment 14:

In embodiment 14, we have reported the partly evaluation of upright withered chain lattice spore cytochrome b gene sequence.Upright withered chain lattice spore is the pathogenic agent of tomato and target.

Collect infected blade material (tomato or potato) from the field, take out fungal material and artificial medium upload be commissioned to train foster.The fungi sample increases by shake-flask culture.The strain isolated at culture collection center is stored in the liquid nitrogen, and regularly artificial medium upload be commissioned to train foster, or before being stored in-80 ℃ again by the host material of the living cultivation of going down to posterity.Grind with the mycelium that will in shaking bottle, grow through the aseptic pestle and the mortar of acid elution, and 100mg is used for genomic dna preparation and the first chain cDNA synthetic (described in step such as the previous embodiment).With every kind of genome diluent of 10 μ l and the pure cDNA of 5 μ l template, use primer deg4F and deg3R (referring to the details among the embodiment 3) as pcr amplification.PCR component and condition are as described in example 3 above.By the described PCR product of gel electrophoresis analysis, find the band (about 500bp) of expection size.When using genomic dna or cDNA as template, described PCR product big or small identical, showing in the DNA district that is increased does not have intron.Described PCR product cloning in TA pCR2.1 carrier, and is inserted segmental 5 clones and checked order the correct size of containing of each clone's incident as mentioned above.When analyzing sequencing data, find that described PCR product all is identical in all cases with the associated biomolecule information software, and a kind of new cytochrome b gene sequence that has close homology with other ascomycetes sequence that demonstrates of encoding.In table 3, can find coding G ₁₄₃The tract of 61 Nucleotide of 30 bases in the second bit base upstream and 30 bases in downstream in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).

When with genomic dna that directly infected tomato leaf extracts from the field template as pcr amplification, and when using primer Ery11-12 (relevant details referring to above), a kind of and previous isolating only different sequence of 12bp of withered chain lattice spore sequence of standing has also increased.Though this dna sequence dna contains the difference of 12 Nucleotide, during with these two kinds of sequence translations, it does not produce any difference on amino acid levels.Significant difference between these two kinds of sequences is at G ₁₄₃There is the intron of an about 1.2kb in 62bp place, the second bit base downstream in the codon.These differences of intron/exon group structure and codon selection aspect prove that these variations are possible in species.

Embodiment 15:

In embodiment 15, we have reported the evaluation of part Semen arachidis hypogaeae tail spore cytochrome b gene sequence.Semen arachidis hypogaeae tail spore is the pathogenic agent of Folium Arachidis hypogaeae spot blight.

Collect infected blade material (peanut) from the field, take out fungal material and artificial medium upload be commissioned to train foster.Culture is stored in the liquid nitrogen stand-by.From store thing, take out material, and on agar, cultivate, with the bacterium colony of shake-flask culture amplification gained, and it is stored in-80 ℃ stand-by.With aseptic pestle and mortar mycelium is ground, and 100mg is used for genomic dna preparation and the first chain cDNA synthetic (described in step such as the previous embodiment) through acid elution.With every kind of genome diluent of 10 μ l and the pure cDNA of 5 μ l template, use primer deg4F and deg3R (referring to the details among the embodiment 3) as pcr amplification.PCR component and condition are as described in example 3 above.By the described PCR product of gel electrophoresis analysis, find the band (about 500bp) of expection size.When using genomic dna or cDNA as template, the size of described PCR product is identical once more, and showing in the DNA district that is increased does not have intron.Described PCR product cloning in TA pCR2.1 carrier, and is inserted segmental 5 clones and checked order the correct size of containing of each clone's incident as previously mentioned.When analyzing sequencing data, find that described PCR product all is identical in all cases with the associated biomolecule information software, and a kind of new cytochrome b gene sequence that has close homology with other cytochrome b gene sequence that demonstrates of encoding.In table 3, can find coding G ₁₄₃The tract of 61 Nucleotide of 30 bases in the second bit base upstream and 30 bases in downstream in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).

Embodiment 16:

In embodiment 16, we have reported the evaluation of part dry thread Pyrenomycetes cytochrome b gene sequence.Dry thread Pyrenomycetes is the pathogenic agent of root stem rot or samping off.

Collect infected blade material (paddy rice) from the field, take out fungal material and artificial medium upload be commissioned to train foster.Culture is stored in the liquid nitrogen stand-by.From store thing, take out material, and on agar, cultivate, with the bacterium colony of shake-flask culture amplification gained, and it is stored in-80 ℃ stand-by.From the isolating RNA of 100mg ground mycelium, it is synthetic to carry out the first chain cDNA as previously mentioned, and 5 μ l are used for pcr amplification, use basidiomycetes degenerated primer 1F (5 ' WYTRGTAYTAATGATGGCTATHGG3 ') (SEQ ID NO 174) and 1R (5 ' TCTTARWATWGCATAGAAWGG3 ') (SEQ ID NO 175), described primer has been described the profile according to the amino acid/11 21-283 of fungal cell's pigment b of yeast saccharomyces cerevisiae numbering system.PCR component and condition are as discussed previously.When by the described PCR product of gel electrophoresis analysis, finds a kind of big or small PCR product (about 500bp) of expecting.With this PCR product cloning, and as previously mentioned 5 clones that contain correct size insertion fragment (about 500bp) are checked order.When analyzing sequencing data, find a kind of new cytochrome b gene sequence that has close homology with other basidiomycetes sequence that demonstrates of described PCR product coding with the associated biomolecule information software.In table 3, can find coding G ₁₄₃The tract of 61 Nucleotide of 30 bases in the second bit base upstream and 30 bases in downstream in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).

Embodiment 17:

In embodiment 17, we have reported the evaluation of the rotten mould cytochrome b gene sequence of part melon and fruit.The melon and fruit corruption is mould to be the pathogenic agent of samping off.

Collect infected blade material (lawn) from the field, take out fungal material and artificial medium upload be commissioned to train foster.Culture is stored in the liquid nitrogen stand-by.From store thing, take out material, and on agar, cultivate, with the bacterium colony of shake-flask culture amplification gained, and it is stored in-80 ℃ stand-by.As described in example 1 above, on 200mg ground mycelium, carry out the genomic dna preparation.By the output of gel electrophoresis analysis genomic dna, storing solution was diluted with 1: 10 and 1: 100 in redistilled water.With the template of every kind of diluent of 10 μ l, use primer PLAS17F and PLAS15R (referring to the details among the embodiment 1) as pcr amplification.PCR component and condition are as described in example 1 above.By the described PCR product of gel electrophoresis analysis, find the band (about 500bp) of a correct size.This PCR product cloning in TA Invitrogen pCR2.1 carrier, and is inserted segmental 5 clones and checked order containing correct size as previously mentioned.When analyzing described sequencing data, find a kind of new cytochrome b gene sequence that close homology is arranged with other oomycetes sequence that demonstrates of described PCR fragment coding with the associated biomolecule information software.In table 3, can find coding G ₁₄₃The tract of 61 Nucleotide of 30 bases in the second bit base upstream and 30 bases in downstream in the codon (according to yeast saccharomyces cerevisiae amino acid numbering system).

Embodiment 18:

In embodiment 18, we have reported the evaluation of part Mycosphaerella musicola cytochrome b gene sequence.M.musicola is the sick pathogenic agent of banana tikka (yellow sigatoka).

Collect infected banana blade from the field, and with on the artificial medium of thecaspore from the blade direct inoculation to culture dish.List cystospore strain isolated maintains on the artificial medium, and by carry out shake-flask culture in broth culture, prepares to be used for pcr analysis.Collect mycelium by Miracloth, and use through the aseptic pestle and the mortar of acid elution and wear into fine powder.100mg ground mycelium is used for extracting genome DNA and the first chain cDNA synthetic (as described in example 7 above).Genomic dna diluted with 1: 10,1: 100 and 1: 1000 in redistilled water as before the pcr template.With 5 μ l cDNA and every kind of genome diluent of 10 μ l template, use degenerated primer to F4/R3 (described in embodiment 3) as pcr amplification.Described in PCR condition and component such as the previous embodiment.During by the described PCR product of gel electrophoresis analysis, when genomic dna and cDNA are used as template, find the band (about 500bp) of an expection size.These two kinds of PCR product cloning in the pCR2.1TA carrier, and are checked order to 5 institute's DCRP that have correct size and insert fragment (about 500bp) with M13 forward primer and reverse primer.During with the relevant suitable described sequence data of bioinformation software analysis, finding that described PCR fragment coding is a kind of has the new cytochrome b gene sequence of close homology with other ascomycetes cytochrome b gene sequence.In table 3, can find coding G ₁₄₃The tract of 61 Nucleotide of 30 bases in the second bit base upstream and 30 bases in downstream in the codon (according to described amino acid numbering system).

<110> was Nika Ltd. <120> method <130> P50396 <150> GB 9910100.8 <151> 1999-04-30 <150> GB 0006004.6 <151> 2000-03-13 <150> GB 0007901.2 <151> 2000-03-31 <160> 196 <170> PatentIn Ver.2.1 <210> 1 <211> 61 <212> DNA <213> Plasmopara viticola (Plasmopara viticola) <400> 1 ttttgccttg gggacaaatg agtttttggg gtgcaacagt tattacaaat ttattctcgg 60 c 61 <210> 2 <211> 61 <212> DNA <213> Erysiphe graminis wheat / barley pathogenic variant (Erysiphe graminis f.sp.tritici / hordei) <400> 2 tattgccata cgggcagatg agccactggg gtgcaaccgt tatcactaac ctaatgagcg 60 c 61 <210> 3 <211> 61 <212> DNA <213> rye beak spore (Rhynchosporium secalis) <400> 3 tgcttcctta tggacagatg tctttatgag gtgccacagt tataactaat cttatgagtg 60 c 61 <210> 4 <211> 61 <212> DNA <213> Round Pyrenophora (Pyrenophora teres) <400> 4 ttttacccta cgggcaaatg agcctttgag gtgctacagt tattactaac cttatgagtg 60 c 61 <210> 5 <211> 61 <212> DNA <213> Solid Pyrenophora (Pyrenophora teres) <400> 5 ttttacccta cgggcaaatg agcctttgag gtgaaatatt tgcctcaaat gtataactaa 60 t 61 <210> 6 <211> 61 <212> DNA <213> He Sheng ball cavity bacteria (Mycosphaerella graminicola) <400> 6 tattacctta tggtcaaatg tctttatgag gagcaacagt tataactaac ttattgagtg 60 c 61 <210> 7 <211> 61 <212> DNA <213> Mycosphaerella fijiensis <400> 7 ttttacctta tggtcaaatg tctttatgag gagctacagt tataactaat ttaatgagcg 60 c 61 <210> 8 <211> 61 <212> DNA <213> monofilament shell (Sphaerothecg fuliginea) <400> 8 tacttccctt cggtcaaatg tcgctctggg gtgcaaccgt tattactaac cttatgagcg 60 c 61 <210> 9 <211> 37 <212> DNA <213> monofilament shell (Sphaerotheca fuliginea) <400> 9 tctggggtgc aaccgttaag taatagcggt tgtaaaa 37 <210> 10 <211> 61 <212> DNA <213> Grapes snagging shell (Uncinula necator) <400> 10 ttttacccta cgggcagatg agcctatggg gtgcaaccgt tattactaac cttatgagcg 60 c 61 <210> 11 <211> 41 <212> DNA <213> Grapes snagging shell (Uncinula necator) <400> 11 agcctatggg gtgcaaccgt taagtaggta atagcggttg a 41 <210> 12 <211> 61 <212> DNA <213> He Sheng stab plate spore (Colletotrichum graminicola) <400> 12 ttttacctta cggacaaatg tcattatgag gtgctacagt tattactaac cttataagtg 60 c 61 <210> 13 <211> 61 <212> DNA <213> Pythium (Pythium aphanidermatum) <400> 13 tattaccttg gggtcaaatg agtttttggg gtgctactgt tattactaat ttattttcag 60 c 61 <210> 14 <211> 61 <212> DNA <213> long spore-like spines disc tray spore (Colletotrichum gloeosporioides) <400> 14 ttttacctta tggacaaatg tcattatgag gtgcaacagt tattactaac cttataagtg 60 c 61 <210> 15 <211> 61 <212> DNA <213> tomato powder spore (Oidium lycopersicum) <400> 15 ttttacccta cgggcagatg agcctgtggg gtgcaaccgt tattactaac cttatgagcg 60 c 61 <210> 16 <211> 61 <212> DNA <213> Tatars within the wire powdery mildew (Leveillula taurica) <400> 16 ttttaccata cggacaaatg tcattatgag gtgcaacagt tattactaac cttatgagtg 60 c 61 <210> 17 <211> 61 <212> DNA <213> Cuban false downy mildew (Pseudoperonospora cubensis) <400> 17 ttttaccttg gggacaaatg agtttttggg gtgcaactgt tattactaat ttattttctg 60 c 61 <210> 18 <211> 61 <212> DNA <213> standing dead Alternaria (Alternaria solani) <400> 18 ttcttcctta tgggcaaatg tctttatgag gtgctacagt tattactaac cttatgagtg 60 c 61 <210> 19 <211> 61 <212> DNA <213> Peanut Cercospora (Cercospora arachidola) <400> 19 tattacctta tggacaaatg tcattatgag gagctacagt tattactaat ttattatctg 60 c 61 <210> 20 <211> 61 <212> DNA <213> Rhizoctonia solani (Rhizoctonia solani) <400> 20 tgcttccata cgggcaaatg tctctgtggg gtgctacagt aattactaat ttactttctg 60 c 61 <210> 21 <211> 61 <212> DNA <213> Mycosphaerella musicola <400> 21 ttttacctta tggtcaaatg tctttatgag gagctacagt tataactaat ttaatgagtg 60 c 61 <210> 22 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 22 ccttggtgac aaatgagttt ttggac 26 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 23 ccatacgggc agatgagcca ctggac 26 <210> 24 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 24 ccttatggac agatgtcttt atgatc 26 <210> 25 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 25 ccctacgggc aaatgagcct ttgcgc 26 <210> 26 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 26 ccttatggtc aaatgtcttt atgaac 26 <210> 27 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 27 ccttatggtc aaatgtcttt atgatc 26 <210> 28 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 28 cccttcggtc aaatgtcgct ctggac 26 <210> 29 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 29 ccctacgggc agatgagcct atggtc 26 <210> 30 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 30 ccttacggac aaatgtcatt atgaac 26 <210> 31 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 31 ccttggtgtc aaatgagttt ttggac 26 <210> 32 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 32 ccttatggac aaatgtcatt atgaac 26 <210> 33 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 33 ccctacgggc agatgagcct gtggac 26 <210> 34 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 34 ccatacggac aaatgtcatt atgaac 26 <210> 35 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 35 ccttggggac aaatgagttt ttggac 26 <210> 36 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 36 ccttatgggc aaatgtcttt atgaac 26 <210> 37 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 37 ccttatggac aaatgtcatt atgaac 26 <210> 38 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 38 ccatacgggc aaatgtctct gtggac 26 <210> 39 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 39 gtgtatggtc aaatgagcct atggcc 26 <210> 40 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 40 ccttatggac agatgtcatt atgaac 26 <210> 41 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 41 ccttggggac aaatgagttt ttggac 26 <210> 42 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 42 ccttatggtc aaatgtcttt atgatc 26 <210> 43 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 43 ccttggtgac aaatgagttt ttggag 26 <210> 44 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 44 ccatacgggc agatgagcca ctggag 26 <210> 45 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 45 ccttatggac agatgtcttt atgatg 26 <210> 46 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 46 ccctacgggc aaatgagcct ttgaag 26 <210> 47 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 47 ccttatggtc aaatgtcttt atgaag 26 <210> 48 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 48 ccttatggtc aaatgtcttt atgatg 26 <210> 49 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 49 cccttcggtc aaatgtcgct ctggag 26 <210> 50 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 50 ccctacgggc agatgagcct atggtg 26 <210> 51 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 51 ccttacggac aaatgtcatt atgaag 26 <210> 52 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 52 ccttggtgtc aaatgagttt ttggag 26 <210> 53 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 53 ccttatggac aaatgtcatt atgaag 26 <210> 54 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 54 ccctacgggc agatgagcct gtggag 26 <210> 55 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 55 ccatacggac aaatgtcatt atgaag 26 <210> 56 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 56 ccttggggac aaatgagttt ttggag 26 <210> 57 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 57 ccttatgggc aaatgtcttt atgaag 26 <210> 58 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 58 ccttatggac aaatgtcatt atgaag 26 <210> 59 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 59 ccatacgggc aaatgtctct gtggag 26 <210> 60 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 60 gtgtatggtc aaatgagcct atggag 26 <210> 61 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 61 ccttatggac agatgtcatt atgaag 26 <210> 62 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 62 ccttggggac aaatgagttt ttggag 26 <210> 63 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 63 ccttatggtc aaatgtcttt atgatg 26 <210> 64 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 64 gatacctaat ggattatttg aacctacct 29 <210> 65 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 65 aacacctaaa ggattaccag atcctgcac 29 <210> 66 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 66 tacacctaaa ggattacctg accctgcac 29 <210> 67 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 67 ttcaagtaca tccaatttca atatacact 29 <210> 68 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 68 taacagaaaa tccacctcat acgaattcaa ctatgtcttg 40 <210> 69 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 69 aaacctcctc aaataaactc aactatatc 29 <210> 70 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 70 taactgagaa accccctcag agaaactcca caatatcttg 40 <210> 71 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 71 ttacagaaaa accacctcaa agaaactcca cgatatcttg 40 <210> 72 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 72 taactgagaa acctcctcaa acgaattcaa caatatcttg 40 <210> 73 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 73 ctacagcaaa tcccccccat aaccaatcaa caatatcttt 40 <210> 74 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 74 taacagagaa acctcctcaa acgaattcaa ctatatcttg 40 <210> 75 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 75 ttacagaaaa acctcctcaa agaaactcca cgatatcttg 40 <210> 76 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 76 ttacagagaa acctcctcaa ataaattcaa ctatatcttg 40 <210> 77 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 77 ctacagcaaa accgccccac aaccaatcaa caatatcttt 40 <210> 78 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 78 taacactgaa acctcctcaa atgaactcaa caatatcttg 40 <210> 79 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 79 aaacagagaa acctcctcat ataaattcaa ctaaatcttg 40 <210> 80 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 80 acacggaaaa gccaccccag attaactcta caaaatcttg 40 <210> 81 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 81 attgacttaa gcctccccac agaaattcga ctatatcttg 40 <210> 82 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 82 taacagaaaa accacctcaa atgaattcaa caatatcttg 40 <210> 83 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 83 caacagcaaa acctccccat aaccaatcaa caatatcttt 40 <210> 84 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 84 taacagaaaa cccacctcaa ataaattcaa ctatatcttg 40 <210> 85 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 85 gccttgggga caaatgagtt tttg 24 <210> 86 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 86 gccatacggg cagatgagcc actg 24 <210> 87 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 87 tccttatgga cagatgtctt tatg 24 <210> 88 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 88 accctacggg caaatgagcc tttgag 26 <210> 89 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 89 taccttatgg tcaaatgtct ttatga 26 <210> 90 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 90 gttttacctt atggtcaaat gtctttatg 29 <210> 91 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 91 ttcccttcgg tcaaatgtcg ctctgg 26 <210> 92 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 92 taccctacgg gcagatgagc ctatgg 26 <210> 93 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 93 taccttacgg acaaatgtca ttatga 26 <210> 94 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 94 taccttgggg tcaaatgagt ttttgg 26 <210> 95 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 95 taccttatgg acaaatgtca ttatga 26 <210> 96 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 96 taccctacgg gcagatgagc ctgtgg 26 <210> 97 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 97 taccatacgg acaaatgtca ttatga 26 <210> 98 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 98 taccttgggg acaaatgagt ttttgg 26 <210> 99 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 99 ttccttatgg gcaaatgtct ttatga 26 <210> 100 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 100 taccttatgg acaaatgtca ttatga 26 <210> 101 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 101 ttccatacgg gcaaatgtct ctgtgg 26 <210> 102 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 102 acgtgtatgg tcaaatgagc ctatgg 26 <210> 103 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 103 taccttatgg acagatgtca ttatga 26 <210> 104 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 104 taccttgggg acaaatgagt ttttgg 26 <210> 105 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 105 taccttatgg tcaaatgtct ttatga 26 <210> 106 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 106 tgaacatatt atgagagatg t 21 <210> 107 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 107 aattgcataa aaaggtaaaa a 21 <210> 108 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 108 aaataacggt tggttaattc g 21 <210> 109 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 109 tcttaaaatt gcataaaaag g 21 <210> 110 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 110 ccttggtgac aaatgagttt ttgtgg 26 <210> 111 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 111 ccttggtgac aaatgagttt ttggag 26 <210> 112 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 112 ccttggtgac aaatgagttt ttggcc 26 <210> 113 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 113 ccttggtgac aaatgagttt ttggac 26 <210> 114 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 114 gccttgggga caaatgagtt tttg 24 <210> 115 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 115 cccgccgtaa ttgtaggggc tgtactaata cggcggg 37 <210> 116 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 116 gatacctaat ggattatttg aacctacct 29 <210> 117 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 117 cccgccctgg gatagccgag aataaatggg cggg 34 <210> 118 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 118 ccttggtgac aaatgagttt ttggag 26 <210> 119 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 119 cccgccctgg gatagccgag aataaatggg cggg 34 <210> 120 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 120 ccttggtgac aaatgagttt ttgagc 26 <210> 121 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 121 cataaccagt caacaacttc ttttcc 26 <210> 122 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 122 cagcttcagc tttcttct 18 <210> 123 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 123 acttaaaggt ctaaattg 18 <210> 124 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 124 atgaacaatt ggtacagtaa t 21 <210> 125 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 125 gttaggtata gatcttaata t 21 <210> 126 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 126 ccatacgggc agatgagcca ctggag 26 <210> 127 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 127 ccatacgggc agatgagcca ctggac 26 <210> 128 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 128 gccatacggg cagatgagcc actg 24 <210> 129 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 129 cccgccgttt tagctgcttt agctttaatg cggcggg 37 <210> 130 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 130 aacacctaaa ggattaccag atcctgcac 29 <210> 131 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 131 aggtytrtay tryggdtcwt a 21 <210> 132 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 132 agcdataacw cctaataatt t 21 <210> 133 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 133 ccttatggac agatgtcttt atgatg 26 <210> 134 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 134 ccttatggac agatgtcttt atgaag 26 <210> 135 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 135 ccttatggac agatgtcttt atgatc 26 <210> 136 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 136 ccttatggac agatgtcttt atgaac 26 <210> 137 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 137 tccttatgga cagatgtctt tatg 24 <210> 138 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 138 cccgccatat tagctgcatt agtat taatg cggcggg 37 <210> 139 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 139 tacacctaaa ggattacctg accctgcac 29 <210> 140 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 140 cttacatctg taataggtaa t 21 <210> 141 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 141 tgttacttta gcaatgcact a 21 <210> 142 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 142 ccctacgggc aaatgagcct ttgaag 26 <210> 143 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 143 ccctacgggc aaatgagcct ttgatc 26 <210> 144 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 144 accctacggg caaatgagcc tttg 24 <210> 145 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 145 tacacctaaa ggatttcctg accctgcaa 29 <210> 146 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 146 acatagtaat actgcttcag c 21 <210> 147 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 147 tacatttgag gcaaatattt c 21 <210> 148 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 148 ctacgggcaa atgagccttt g 21 <210> 149 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 149 ctctggaact atcgctgcag g 21 <210> 150 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 150 cccgccgcaa gctgatttca taggcggg 28 <210> 151 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 151 ttcaagtaca tccaatttca atatacact 29 <210> 152 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 152 accctacggg caaatgagcc tttgag 26 <210> 153 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 153 ccctacgggc aaatgagcct ttgaag 26 <210> 154 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 154 ccctacgggc aaatgagcct ttgaac 26 <210> 155 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 155 ccctacgggc aaatgagcct ttgacg 26 <210> 156 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 156 ccctacgggc aaatgagcct ttgacc 26 <210> 157 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 157 ccctacgggc aaatgagcct tcgaag 26 <210> 158 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 158 ccctacgggc aaatgagcct tcgaac 26 <210> 159 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 159 ccctacgggc aaatgagcct tttacg 26 <210> 160 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 160 ccctacgggc aaatgagcct tttacc 26 <210> 161 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 161 ccctacgggc aaatgagcct ttgcgg 26 <210> 162 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 162 ccctacgggc aaatgagcct ttgcgc 26 <210> 163 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 163 atgaacaatt ggtacagtaa t 21 <210> 164 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 164 aaatctgtta aaggcatagc c 21 <210> 165 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 165 gttttaccct acgggcagat g 21 <210> 166 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 166 aaagaatctg tttaaggttg c 21 <210> 167 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 167 aaaccacctc aaagaaactc c 21 <210> 168 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 168 catgaatagg acaagatatc g 21 <210> 169 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 169 tcacctagaa cattaacatg a 21 <210> 170 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 170 aaatctgtta aaggcatagc c 21 <210> 171 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 171 ttcccttcgg tcaaatgtcg c 21 <210> 172 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 172 aaaccccctc agagaaactc c 21 <210> 173 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 173 gaccccgcgc tatcatgtaa g 21 <210> 174 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 174 wytrgtayta atgatggcta thgg 24 <210> 175 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 175 tcttarwatw gcatagaawg g 21 <210> 176 <211> 61 <212> DNA <213> Plasmopara viticola (Plasmopara viticola) <400> 176 ttttgccttg gggacaaatg agtttttggg ctgcaacagt tattacaaat ttattctcgg 60 c 61 <210> 177 <211> 61 <212> DNA <213> Erysiphe graminis wheat / barley pathogenic variant (Erysiphe graminis f.sp.tritici / hordei) <400> 177 tattgccata cgggcagatg agccactggg ctgcaaccgt tatcactaac ctaatgagcg 60 c 61 <210> 178 <211> 61 <212> DNA <213> rye beak spore (Rhynchosporium secalis) <400> 178 tgcttcctta tggacagatg tctttatgag ctgccacagt tataactaat cttatgagtg 60 c 61 <210> 179 <211> 61 <212> DNA <213> Round Pyrenophora (Pyrenophora teres) <400> 179 ttttacccta cgggcaaatg agcctttgag ctgctacagt tattactaac cttatgagtg 60 c 61 <210> 180 <211> 61 <212> DNA <213> Round Pyrenophora (Pyrenophora teres) <400> 180 ttttacccta cgggcaaatg agcctttgag ctgaaatatt tgcctcaaat gtataactaa 60 t 61 <210> 181 <211> 61 <212> DNA <213> He Sheng ball cavity bacteria (Mycosphaerella graminicola) <400> 181 tattacctta tggtcaaatg tctttatgag cagcaacagt tataactaac ttattgagtg 60 c 61 <210> 182 <211> 61 <212> DNA <213> Mycosphaerella fijiensis <400> 182 ttttacctta tggtcaaatg tctttatgag cagctacagt tataactaat ttaatgagcg 60 c 61 <210> 183 <211> 61 <212> DNA <213> monofilament shell (Sphaerotheca fuliginea) <400> 183 tacttccctt cggtcaaatg tcgctctggg ctgcaaccgt tattactaac cttatgagcg 60 c 61 <210> 184 <211> 37 <212> DNA <213> monofilament shell (Sphaerotheca fuliginea) <400> 184 tctgggctgc aaccgttaag taatagcggt tgtaaaa 37 <210> 185 <211> 61 <212> DNA <213> Grapes snagging shell (Uncinula necator) <400> 185 ttttacccta cgggcagatg agcctatggg ctgcaaccgt tattactaac cttatgagcg 60 c 61 <210> 186 <211> 41 <212> DNA <213> Grapes snagging shell (Uncinula necator) <400> 186 agcctatggg ctgcaaccgt taagtaggta atagcggttg a 41 <210> 187 <211> 61 <212> DNA <213> He Sheng stab plate spore (Colletotrichum graminicola) <400> 187 ttttacctta cggacaaatg tcattatgag ctgctacagt tattactaac cttataagtg 60 c 61 <210> 188 <211> 61 <212> DNA <213> Pythium (Pythium aphanidermatum) <400> 188 tattaccttg gggtcaaatg agtttttggg ctgctactgt tattactaat ttattttcag 60 c 61 <210> 189 <211> 61 <212> DNA <213> long spore-like spines disc tray spore (Colletotrichum gloeosporioides) <400> 189 ttttacctta tggacaaatg tcattatgag ctgcaacagt tattactaac cttataagtg 60 c 61 <210> 190 <211> 61 <212> DNA <213> tomato powder spore (Oidium lycopersicum) <400> 190 ttttacccta cgggcagatg agcctgtggg ctgcaaccgt tattactaac cttatgagcg 60 <210> 191 <211> 61 <212> DNA <400> 191 <210> 192 <211> 61 <212> DNA <400> 192 <210> 193 <211> 61 <212> DNA <400> 193 <210> 194 <211> 61 <212> DNA <400> 194 <210> 195 <211> 61 <212> DNA <400> 195 <211> 61 <212> DNA <400> 196 ...

Claims (53)

1. one kind is used for detecting the method that fungal nucleic acid suddenlys change, the existence of wherein said sudden change causes the resistance of fungi to other any compound in strobilurins analogue or the same crossed resistance group, and described method comprises with arbitrary (or a kind of) mononucleotide polymorphic detection technique identifying whether there is described sudden change in fungal nucleic acid.

2. one kind is used for detecting the method that fungal nucleic acid suddenlys change, the existence of wherein said sudden change causes the resistance of fungi to any other compound in strobilurins analogue or the same crossed resistance group, described method comprises the existence of the amplicon that detection produces between the reaction period at PCR, wherein said PCR reaction is included in suitable Nucleotide triphosphoric acid and is used for polymeric reagent and exists down, the given the test agent that comprises fungal nucleic acid is contacted with a kind of diagnostic primer, whether exist described sudden change directly related in the detection of wherein said amplicon and the described nucleic acid.

3. one kind is used for detecting the method that fungal nucleic acid suddenlys change, the existence of wherein said sudden change causes the resistance of fungi to any other compound in strobilurins analogue or the same crossed resistance group, described method comprises: at suitable Nucleotide triphosphoric acid and being used in the presence of the polymeric reagent, the given the test agent that comprises fungal nucleic acid is contacted with a kind of suitable diagnostic primer, make and perhaps when having wild-type sequence, extended described diagnostic primer or extended when in described sample, having described sudden change; And whether exist according to the diagnostic primer extension product and to detect described sudden change and whether exist.

4. a claim 2 is used for detecting the method that fungal nucleic acid suddenlys change, wherein said method comprises: at suitable Nucleotide triphosphoric acid and being used in the presence of the polymeric reagent, the given the test agent that comprises fungal nucleic acid is contacted with the diagnostic primer of described specific sudden change, make described diagnostic primer be extended when in described sample, having described sudden change; And whether exist according to the diagnostic primer extension product and to detect described sudden change and whether exist.

5. the method for an arbitrary aforementioned claim, wherein in fungal cell's pigment b gene, there is described sudden change, wherein said sudden change causes any other compound in strobilurins analogue or the same crossed resistance group for the inhibition in described cytochrome b protein-active site, but still allows respiratory to take place.

6. method according to aforementioned claim, the sudden change in the wherein said fungal nucleic acid cause in coded protein with brewing yeast cell pigment b residue 143 corresponding positions on glycine residue be selected from following a kind of aminoacid replacement: arginine, Serine, halfcystine, Xie Ansuan, aspartic acid and L-Ala.

7. the method for an arbitrary aforementioned claim, the sudden change in the wherein said fungal nucleic acid cause in coded protein with brewing yeast cell pigment b residue 143 corresponding positions on glycine residue replaced by L-Ala.

8. the method for a claim 2, described method is used for detecting fungal cell's pigment b gene and causes G in the coded protein ₁₄₃The sudden change that A replaces, wherein said sudden change causes the resistance of fungi to any other compound in strobilurins analogue or the same crossed resistance group, described method comprises the existence of the amplicon that detection PCR produced between the reaction period, wherein said PCR reaction is included in suitable Nucleotide triphosphoric acid and a kind of polymeric reagent that is used for exists down, the given the test agent that comprises fungal nucleic acid is contacted with a kind of primer, whether exist described sudden change directly related in the detection of wherein said amplicon and the described nucleic acid.

9. the method for a claim 3, described method is used for detecting fungal cell's pigment b gene and causes G in the coded protein ₁₄₃The sudden change that A replaces, described method comprises: in suitable Nucleotide triphosphoric acid and a kind of being used in the presence of the polymeric reagent, make the given the test agent that comprises fungal nucleic acid and for causing G in the coded protein ₁₄₃The diagnostic primer contact of the sudden change that A replaces makes described diagnostic primer exist in described sample and causes G in the coded protein ₁₄₃Extended during sudden change that A replaces; And whether exist according to the diagnostic primer extension product and to detect described sudden change and whether exist.

10. the method for an arbitrary aforementioned claim, wherein said fungal gene are present in and are selected from following a kind of phytopathogenic fungi: grape is given birth to single shaft mould (Plasmopara viticola), the mutation (Erysiphe graminis f.sp.tritici/hordei) of causing a disease of standing grain powdery mildew wheat/barley, rye beak spore (Rhynchosporium secalis), circle nuclear cavity bacteria (Pyrenophora teres), standing grain green-ball chamber bacterium (Mycosphaerella graminicola), venturia inaequalis (Venturiainaequalis), Mycosphaerella fijiensis var.difformis, monofilament shell (Sphaerotheca fuliginea), grape snag shell (Uncinula necator), standing grain is given birth to thorn dish spore (Colletotrichum graminicola), melon and fruit corruption mould (Pythium aphanidermatum), Colletotrichum gloeosporiodes (Colletotrichum gloeosporiodes), tomato powder spore (Oidiumlycopersicum), Magnaporthe grisea, phytophthora infestans (Phytophthorainfestans), Tartar's internal thread powdery mildew (Leveillula taurica), the false downy mildew (Pseudoperonospora cubensis) of Cuba, upright withered chain lattice spore (Alternaria solani), dry thread Pyrenomycetes (Rhizoctonia solani), Mycosphaerella musicola and Semen arachidis hypogaeae tail spore (Cercospora arachidola).

11. one kind is used for detecting the method for fungi to the resistance of any other compound of strobilurins analogue or same crossed resistance group, described method comprises identifying in the fungal nucleic acid whether have sudden change, the existence of wherein said sudden change causes the resistance to any other compound in strobilurins analogue or the same crossed resistance group, described method comprise identify occur in the described cytochrome b albumen of coding with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in whether have single nucleotide polymorphism on the correspondence position of one or more bases.

12. the method for a claim 11, wherein said single nucleotide polymorphism occur in the amino acid whose triplet on coding and brewing yeast cell pigment b residue 143 corresponding positions on the correspondence position of second bit base.

13. the method for an arbitrary aforementioned claim, wherein the single nucleotide polymorphism that is detected be in the described cytochrome b albumen of coding with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in the G base to take place on the correspondence position of second bit base become the C base.

14. proteic fungal DNA sequence of all or part of cytochrome b of coding, wherein said dna sequence dna with brewing yeast cell pigment b residue 143 corresponding positions on the glycine residue of encoding, and described dna sequence dna can derive from and is selected from following a kind of fungi: it is mould that grape is given birth to single shaft, the mutation of causing a disease of standing grain powdery mildew wheat/barley, rye beak spore, the circle nuclear cavity bacteria, standing grain green-ball chamber bacterium, Mycosphaerella fijiensis var.difformis, the monofilament shell, grape snag shell, standing grain is given birth to thorn dish spore, the melon and fruit corruption is mould, Colletotrichum gloeosporiodes, the tomato powder spore, Tartar's internal thread powdery mildew, the false downy mildew of Cuba, upright withered chain lattice spore, dry thread Pyrenomycetes, Mycosphaerella musicola and Semen arachidis hypogaeae tail spore.

15. the fungal DNA sequence of a claim 14, described fungal DNA sequence comprises full sequence or the partial sequence that is selected from following a kind of dna sequence dna: SEQ ID NO1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQID NO 10, SEQ ID NO11, SEQ ID NO 12, SEQ ID NO 13, SEQID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQID NO 18, SEQ ID NO 19, SEQ ID NO 20 and SEQ ID NO 21.

16. proteic fungal DNA sequence of all or part of cytochrome b of coding, when described sequence is alignd arrangement on the proteic corresponding wild-type dna sequence dna of Codocyte pigment b, can see described fungal DNA sequence in described DNA in the encoding said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in contain the single nucleotide polymorphism sudden change on the correspondence position of one or more bases, described single nucleotide polymorphism sudden change causes described normal glycine residue by a kind of alternate aminoacid replacement, and prerequisite is that described dna sequence dna is not the cytochrome b encoding sequence of the little mushroom of newborn handle.

17. the fungal DNA sequence of a claim 16, the sudden change of wherein said single nucleotide polymorphism occur in the code for said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet on the correspondence position of second bit base, described single nucleotide polymorphism sudden change causes described normal glycine residue by a kind of alternate aminoacid replacement, and prerequisite is that described dna sequence dna is not the cytochrome b encoding sequence of the little mushroom of newborn handle.

18. proteic fungal DNA sequence of all or part of cytochrome b of coding, wherein said dna sequence dna with brewing yeast cell pigment b residue 143 corresponding positions on the alanine residue of encoding, and described dna sequence dna can derive from and is selected from following a kind of fungi: it is mould that grape is given birth to single shaft, the mutation of causing a disease of standing grain powdery mildew wheat/barley, rye beak spore, the circle nuclear cavity bacteria, standing grain green-ball chamber bacterium, venturia inaequalis, Mycosphaerella fijiensis var.difformis, the monofilament shell, grape snag shell, standing grain is given birth to thorn dish spore, the melon and fruit corruption is mould, Colletotrichum gloeosporiodes, the tomato powder spore, Magnaporthe grisea, phytophthora infestans, Tartar's internal thread powdery mildew, the false downy mildew of Cuba, upright withered chain lattice spore, dry thread Pyrenomycetes, Mycosphaerella musicola and Semen arachidis hypogaeae tail spore.

19. the proteic fungal DNA sequence of all or part of cytochrome b of the coding of a claim 18, wherein said dna sequence dna contains a single nucleotide polymorphism, described single nucleotide polymorphism cause in encoding said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in the above normal guanine residue of correspondence position of second bit base replaced by the cytosine(Cyt) residue, prerequisite is that described dna sequence dna is not the cytochrome b encoding sequence of the little mushroom of newborn handle.

20. each fungal DNA sequence among the claim 16-19, described fungal DNA comprises full sequence or the partial sequence that is selected from following a kind of sequence: SEQ ID NO176, SEQ ID NO 177, SEQ ID NO 178, SEQ ID NO 179, SEQ IDNO 180, SEQ ID NO 181, SEQ ID NO 182, SEQ ID NO 183, SEQ ID NO 184, SEQ ID NO 185, SEQ ID NO 186, SEQ ID NO187, SEQ ID NO 188, SEQ ID NO 189, SEQ ID NO 190, SEQ IDNO 191, SEQ ID NO 192, SEQ ID NO 193, SEQ ID NO 194, SEQ ID NO 195 and SEQ ID NO 196.

21. give the fungal cell pigment b albumen of fungi for one kind to the resistance of the compound in strobilurins analogue or the same crossed resistance group, wherein owing in the DNA of encoding said proteins, there is a sudden change, and cause that a normal glycine residue is changed in described albumen, described sudden change occur in the encoding said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet on the correspondence position of one or more bases, prerequisite is that described albumen is not the little mushroom cytochrome b of newborn handle albumen.

22. fungal cell's pigment b albumen of a claim 21, wherein owing in the DNA of encoding said proteins, there is a sudden change, and cause that a normal glycine residue is changed in described albumen, described sudden change occur in the encoding said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet on the correspondence position of second bit base, prerequisite is that described albumen is not the little mushroom cytochrome b of newborn handle albumen.

23. cause in fungal cell's pigment b gene in institute's encoding proteins with saccharomyces cerevisiae cytochrome b residue 143 relevant positions on the detection method of the substituted sudden change of glycine residue; Described method comprises identifying in the fungal nucleic acid sample whether have described sudden change, wherein any (or a kind of) mononucleotide polymorphic detection method based on come in comfortable coding or wild-type protein or mutein matter with saccharomyces cerevisiae cytochrome b residue 143 relevant positions on amino acid whose triplet in the about sequence information of 30-90 nucleotides in the upstream of correspondence position of one or more bases and/or downstream.

24. the method for a claim 23, wherein any (or a kind of) mononucleotide polymorphic detection method based on come in comfortable coding or wild-type protein or the mutant protein with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in the sequence information of upstream and/or downstream about 30-90 Nucleotide of correspondence position of second bit base.

25. allele specific oligonucleotide, described oligonucleotide can combine with the proteic fungal nucleic acid sequence of the encoding wild type cytochrome b that is selected from following fungi: it is mould that grape is given birth to single shaft, the mutation of causing a disease of standing grain powdery mildew wheat/barley, rye beak spore, the circle nuclear cavity bacteria, standing grain green-ball chamber bacterium, Mycosphaerella fijiensis var.difformis, the monofilament shell, grape snag shell, standing grain is given birth to thorn dish spore, the melon and fruit corruption is mould, Colletotrichum gloeosporiodes, the tomato powder spore, Tartar's internal thread powdery mildew, the false downy mildew of Cuba, upright withered chain lattice spore, dry thread Pyrenomycetes, Mycosphaerella musicola and Semen arachidis hypogaeae tail spore, wherein said oligonucleotide comprises one section recognition sequence, the identification of described recognition sequence a kind of with brewing yeast cell pigment b residue 143 corresponding positions on the nucleotide sequence of coding glycine residue.

26. allele specific oligonucleotide, described oligonucleotide can combine with the proteic fungal nucleic acid sequence of encoding mutant type cytochrome b, wherein said oligonucleotide comprises one section recognition sequence, the identification of described recognition sequence a kind of with brewing yeast cell pigment b residue 143 corresponding positions on coding be selected from following a kind of amino acid whose nucleotide sequence: arginine, Serine, halfcystine, Xie Ansuan, aspartic acid, L-glutamic acid, tryptophane or L-Ala.

27. diagnostic primer or diagnostic oligonucleotide, described primer or oligonucleotide can combine with the template that comprises mutant fungal cell pigment b nucleotide sequence, the Nucleotide that wherein said primer or oligonucleotide 3 ' last Nucleotide correspondence exist in described mutant fungal cell's pigment b gene, and the existence of described Nucleotide causes the resistance of fungi to any other compound in strobilurins analogue or the same crossed resistance group.

28. the Nucleotide that exists in the diagnostic primer of a claim 27, the penult Nucleotide (2) of wherein said primer or (3) and corresponding position described in the described wild-type cell pigment b sequence is different.

29. be used for detecting and be selected from following fungal cell's pigment b gene G ₁₄₃One or more diagnostic primers of A sudden change: SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO40, SEQ ID NO 41 and SEQ ID NO 42 and their derivative, last Nucleotide of wherein said 3 ' end is identical with the above sequence that provides, and wherein under the situation of the characteristic of the described diagnostic primer of remarkably influenced, 10 at the most of described all the other Nucleotide, for example at the most 8,6,4,2,1 can change.

30. an alleles-specific oligonucleotide probe, described probe can detect in described DNA in the code for said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in fungal cell's pigment b gene pleiomorphism of correspondence position of one or more bases.

31. the alleles-specific oligonucleotide probe of a claim 30, wherein said polymorphic position in described DNA in the code for said proteins with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in the correspondence position of second bit base.

32. being guanine bases, the alleles-specific oligonucleotide probe of a claim 31, wherein said polymorphism change into the cytosine(Cyt) base.

33. a diagnostic kit, described test kit comprise diagnostic primer or the allele specific oligonucleotide of a kind of claim 25 or 26 or alleles-specific oligonucleotide probe, Nucleotide triphosphoric acid, polysaccharase and the damping fluid of a kind of claim 30-32 of one or more claims 27-29.

34. method that detects phytopathogenic fungi to the mycocide resistance, described method comprises that with the sudden change in any (or a kind of) mononucleotide polymorphic technology for detection fungal gene the existence of wherein said sudden change causes for the resistance of its target protein by the mycocide of chondriogen coding.

35. method that detects phytopathogenic fungi to the mycocide resistance, described method comprises the existence of the amplicon that detection produces between the reaction period at PCR, wherein said PCR reaction is included in suitable Nucleotide triphosphoric acid and a kind of polymeric reagent that is used for exists down, the given the test agent that comprises fungal nucleic acid is contacted with a kind of primer, whether exist described sudden change directly related in the detection of wherein said amplicon and the described nucleic acid, the existence of wherein said sudden change causes for the resistance of its target protein by the mycocide of chondriogen coding.

36. the detection phytopathogenic fungi of claim 34 or claim 35 is to the method for mycocide resistance, described method comprises: at suitable Nucleotide triphosphoric acid and being used in the presence of the polymeric reagent, the given the test agent that comprises fungal nucleic acid is contacted with the diagnostic primer that has the specific sudden change that causes the mycocide resistance, make described diagnostic primer be extended when in described sample, having described sudden change; And whether exist according to the diagnostic primer extension product and to detect described sudden change and whether exist.

37. the detection of a mutation frequency and quantivative approach, described sudden change causes phytopathogenic fungi to the resistance of target protein by the mycocide of chondriogen coding, described method comprises: detect in the fungal gene whether have sudden change, the existence of wherein said sudden change causes the resistance of fungi to any other compound in strobilurins analogue or the same crossed resistance group, and described method comprises with any (or a kind of) mononucleotide polymorphic detection technique identifying with quantitative whether there being described sudden change in the fungal nucleic acid.

38. the method for a claim 37, described method comprises the existence of the amplicon that detection produces between the reaction period at PCR, wherein said PCR reaction is included in suitable Nucleotide triphosphoric acid and a kind of polymeric reagent that is used for exists down, the given the test agent that comprises fungal nucleic acid is contacted with a kind of suitable primer, whether exist described sudden change directly related in the detection of wherein said amplicon and the described nucleic acid, the existence of wherein said sudden change causes for the resistance of target protein by the mycocide of chondriogen coding; And according to the amplicon that whether exists described PCR to be produced between the reaction period and to the relative existence of described sudden change with do not exist and detect and quantitatively.

39. the method for claim 37 or claim 38, described method is included in suitable Nucleotide triphosphoric acid and a kind of polymeric reagent that is used for exists down, the given the test agent that comprises fungal nucleic acid is contacted with the diagnostic primer, cause in the described nucleic acid to detect that the specific sudden change by the mycocide resistance of the mycocide of chondriogen coding exists and do not exist for target protein, make and extended when not existing and existing relevant described diagnostic primer in described sample, to have described suitable fungi template with described specific sudden change; And according to whether having the diagnostic primer extension product to the relative existence of described sudden change with do not exist and detect and quantitatively.

40. a selection is applied to effective mycocide and the suitableeest method of using level thereof of crop, described method comprises: analysis can be infected the fungi sample of described crop, and detect and/or quantitatively exist/and do not exist from sudden change in the gene of described fungi, the existence of wherein said sudden change can cause selecting effective mycocide and the suitableeest level of using thereof then for the resistance of target protein by the mycocide of chondriogen coding.

41. the method for a claim 40, wherein said detection method is used any (or a kind of) mononucleotide polymorphic detection technique.

42. the method for claim 40 or claim 41, wherein said detection method comprises: at suitable Nucleotide triphosphoric acid and being used in the presence of the polymeric reagent, the given the test agent that comprises fungal nucleic acid is contacted with the diagnostic primer of described specific sudden change, make described diagnostic primer be extended when in described sample, having described sudden change; And whether exist according to the diagnostic primer extension product and to detect described sudden change and whether exist.

43. a method of preventing and treating the fungi infestation of crop, described method comprise mycocide is applied to described crop, wherein said mycocide is according to each is selected among the claim 40-42.

44. each method among the claim 34-43, wherein said mycocide is any other compound in strobilurins analogue or the same crossed resistance group.

45. one kind is used for detecting the method for fungi to the resistance of any other compound of strobilurins analogue or same crossed resistance group, described method comprises identifying in the fungal nucleic acid whether have sudden change, the existence of wherein said sudden change causes the resistance to any other compound in strobilurins analogue or the same crossed resistance group, described method comprise evaluation whether exist among the described DNA in the described cytochrome b albumen of coding with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in the single nucleotide polymorphism that takes place on the correspondence position of one or more bases.

46. a claim 45 be used to detect the method for fungi to strobilurins analogue resistance, described method comprise evaluation whether exist among the described DNA in the described cytochrome b albumen of coding with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet in a single nucleotide polymorphism taking place on the correspondence position of second bit base.

47. the method for an arbitrary aforementioned claim, wherein said detection and/or quantivative approach are based on the fluoroscopic examination to the diagnostic primer extension product.

48. relating to, the method for an arbitrary aforementioned claim, wherein said detection method use Scorpion ^TMDetection system.

49. each method among the claim 34-48, wherein said sudden change occur among the described DNA in the described cytochrome b albumen of coding with brewing yeast cell pigment b residue 143 corresponding positions on amino acid whose triplet on the correspondence position of second bit base.

50. each method among the claim 34-49, wherein said sudden change is to cause the guanine of G143A replacement in the coded protein to become the change of cytosine(Cyt), wherein with brewing yeast cell pigment b residue 143 corresponding positions on, a wild-type glycine residue is replaced by alanine residue.

51. computer readable medium that stores arbitrary sequence in arbitrary aforementioned claim, described sequence comprises: proteic all or part of dna sequence dna of encoding mutant type cytochrome b as herein described or protein sequence, the existence of wherein said sudden change cause the resistance of fungi to any other compound in strobilurins analogue or the same crossed resistance group; Derive from all or part of dna sequence dna or the protein sequence of the encoding wild type cytochrome b sequence that is selected from following a kind of fungi: it is mould that grape is given birth to single shaft, the mutation of causing a disease of standing grain powdery mildew wheat/barley, rye beak spore, the circle nuclear cavity bacteria, standing grain green-ball chamber bacterium, venturia inaequalis, Mycosphaerella fijiensisvar.difformis, the monofilament shell, grape snag shell, standing grain is given birth to thorn dish spore, the melon and fruit corruption is mould, Colletotrichum gloeosporiodes, the tomato powder spore, Magnaporthe grisea, phytophthora infestans, Tartar's internal thread powdery mildew, the false downy mildew of Cuba, upright withered chain lattice spore, dry thread Pyrenomycetes, Mycosphaerellamusicola and Semen arachidis hypogaeae tail spore; Or according to any allele specific oligonucleotide, allele-specific primers, allele specific oligonucleotide primer, general primer or the diagnostic primer of arbitrary claim.

52. a diagnostic kit, described diagnostic kit are used for claim 1-13,21 or each method of 34-50.

53. the diagnostic kit of a claim 52, described test kit comprise one or more following components: diagnostic Oligonucleolide primers, wild-type Oligonucleolide primers, control oligonucleotide primer and/or shared Oligonucleolide primers, alleles-specific oligonucleotide probe, suitable Nucleotide triphosphoric acid be dATP, dCTP, dGTP, dTTP, a kind of suitable polysaccharase and a kind of damping fluid for example.

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