CN104593480A

CN104593480A - Application of comparative genomics to rapid identification of phaseolus vulgaris mildew resistance locus o gene

Info

Publication number: CN104593480A
Application number: CN201310524705.9A
Authority: CN
Inventors: 钱孝英; 袁伟; 俞锞; 刘云飞; 钱春桃
Original assignee: Jiangsu Changshu Modern Agricultural Industrial Park Development Co Ltd
Current assignee: Jiangsu Changshu Modern Agricultural Industrial Park Development Co Ltd
Priority date: 2013-10-30
Filing date: 2013-10-30
Publication date: 2015-05-06

Abstract

Relating to plant comparative genomics, genetics, bioinformatics and other disciplinary knowledge, the invention belongs to the scientific field of plant biotechnology, and discloses rapid identification of phaseolus vulgaris mildew resistance locus o gene. The main steps include: 1) download of phaseolus vulgaris whole genome sequence and acquisition of MLO type gene; 2) identification of MLO type gene; 3) MLO type gene phylogenetic relationship; and 4) comparison of MLO type powdery mildew. The invention effectively shortens the mining cycle of phaseolus vulgaris powdery mildew gene, and is conducive to rapid identification of powdery mildew gene. Corresponding coseparation functional markers (SNP, SCAR and the like) are developed through identified candidate powdery mildew genes, which also can be used for molecular marker-assisted selection of mildew resistance locus o gene and have high accuracy. Multi-resistance breeding materials can be created by combining other resistant gene molecular markers, the breeding period can be shortened, and the breeding efficiency is improved, thus laying the foundation for elaborating the phaseolus vulgaris mildew resistance molecular mechanism.

Description

Use comparative genomics Rapid identification Kidney bean mildew-resistance gene

Technical field

The present invention is by means of Kidney bean order-checking whole genome sequence, utilize the method Rapid identification bean powdery mildew genes such as plant comparative genomics, genetics, information biology and candidate gene strategy, be mainly concerned with the download of Kidney bean whole genome sequence, the qualification of candidate gene, the comparison of gene, the means such as cluster, and then identify powdery mildew gene, belong to Plant Biotechnology scientific domain.

Background technology

Kidney bean is the main Food Legume crops of the mankind, and accounting for 50% of global Food Legume ultimate production, is one of main source of vegetable-protein.Bean powdery mildew generally occurs in China various places, be produce in Major Diseases, the production loss caused every year up to 30%, even up to 80% time serious.At present aborning, spraying fungicide and to promote disease-resistant variety be main prevention and controls, but life-time service medicine can cause environmental pollution and Biological Strains of The Pest variation, therefore promoting disease-resistant variety is method the most safely and effectively.Current domestic Kidney bean breeding backwardness relatively, the disease-resistant variety used in production is main dependence on import also.The strict selfing crop of Phaseolus, the special tectonic of its floral organ causes general hybridization, backcross etc., and languages means are difficult to carry out on Kidney bean, therefore utilize conventional breeding methods breeding resistant variety to seem very difficult.Excavate disease-resistant gene, utilize biotechnology Innovation Germplasm to become the emphasis of next step people research.Bean powdery mildew belongs to caused by (Oidium spp.) infect by the powder spore of Deuteromycotina.The germ perfect stage returns many capsules silk shell of Ascomycotina to belong to (Erysiphe spp) or single capsule silk shell belongs to (Sphaerotheca spp.), but about the research data of the differentiation of bean powdery mildew bacterial classification and its genetics of resistance rule is less, there is no bibliographical information at present, this seriously inhibits the paces of bean powdery mildew research.

MLO is the distinctive class disease-resistant gene of plant, is first disease-resistant gene that it is found that.The mankind find that MLO gene is in barley the earliest, and people find that this gene does not have function in mildew-resistance kind under study for action, and when making it not play function its silence of plan, this kind can produce resistance to Powdery Mildew.The resistance mechanism of MLO to plant plays negative regulation effect, is equivalent to " susceptible " gene of plant to a certain extent, and the recognition reaction of similar non-host can occur for its sudden change or disappearance, causes the resistance of wide spectrum of plant generation to all known microspecies of pathogenic bacteria.Therefore, this genoid has very large potentiality and application prospect improving in plant resistance to environment stress.Recently, research finds tomato, and the powdery mildew gene of a lot of plant such as pea, Arabidopis thaliana, rose, capsicum, Root or stem of Littleleaf Indianmulberry is all that MLO type disease-resistant gene controls.Therefore the MLO type disease-resistant gene excavated in plant has great importance to Biology of Plant-Powdery Mildew Interaction breeding.Existing research shows, MLO gene in familial form, has multiple family member, wherein has 15 family members in Arabidopis thaliana in many crops.In corn, find to have 9 at least.In paddy rice, there are 12 family members, due to the factor such as delayed that the genome sequencing of other plant is done, barley, the crop such as wheat, lotus rhizome, capsicum, tomato relatively seldom have other member of MLO family to be found.

At present, the method for clone plant disease-resistant gene mainly contains transposon tagging, map based cloning method etc.But because the fundamental research of Kidney bean is not deep enough, therefore utilize these methods to be difficult to clone these genes exactly.Therefore, the MLO type disease-resistant gene how in Rapid identification Kidney bean will become the important prerequisite of Kidney bean mildew-resistance breeding.Recently, completing of bean gene group order-checking provides condition for we excavate bean powdery mildew gene fast.This patent describes premised on Kidney bean whole genome sequence, in conjunction with knowledge such as genetics, genomics and information biology, excavates the method for powdery mildew gene fast.

MLO type disease-resistant gene is the special class disease-resistant gene of plant.Investigator find the earliest MLO ( mildew resistance locus o) gene pairs powder mildew resistance starts from 1937-1938, acquired the barley of a lot of kind in Ethiopia by fritz, two strains wherein all have efficient resistance to Powdery Mildew (Blumeriagraminis f.sp.hordei) all known physiological strains.Further research shows, in barley, the recessive mutation mlo of MLO gene can make barley produce resistance that is lasting, wide spectrum to the physiological strain of nearly all known barley powdery mildew bacteria.Recently, investigator finds that the mildew-resistance gene of a lot of plant is all MLO type Gene Handling, as tomato, and pea, Arabidopis thaliana, rose, capsicum, Root or stem of Littleleaf Indianmulberry etc.Therefore the MLO type disease-resistant gene excavated in plant has important effect to Biology of Plant-Powdery Mildew Interaction breeding.

At present, the conventional method excavating disease-resistant gene conventional has map based cloning, the methods such as transposon tagging.But because the fundamental research of Kidney bean is not deep enough, not only the time is grown but also is difficult to clone these genes exactly therefore to utilize these methods.Therefore, the MLO type disease-resistant gene how in Rapid identification Kidney bean will become the important prerequisite of Kidney bean mildew-resistance breeding.

Plant comparative genomics (Comparative Genomics) is based on Genome Atlas and order-checking basis, compares, understand the subject of the function of gene, expression mechanisms and spore to known gene and genome structure.Between Land use models Plant Genome and other Plant Genome on coded sequence and structural homology, clone's other plant gene, discloses gene function and molecular mechanism, illustrates spore relation and genomic immanent structure.The method that this patent adopts and thinking: the research of model plant arabidopsis gene group has disclosed the function of MLO type gene, utilize the homology clone Kidney bean MLO type disease-resistant gene in its order of gene, according to the feature of the superiority in model plant Arabidopis thaliana experimental system and known MLO type disease-resistant gene, " seizure " Kidney bean mildew-resistance gene fast.This patent describes premised on Kidney bean whole genome sequence, in conjunction with knowledge such as comparative genomics, genetics, genomics, information biology and candidate gene strategy, excavates powdery mildew gene fast.

Summary of the invention

Technical problem

The object of this invention is to provide a kind of by conjunction with knowledge such as plant comparative genomics, plant genetics, genomics and information biology, excavate Kidney bean mildew-resistance gene fast.Its result can be used for the exploitation of bean powdery mildew gene compact linkage molecule mark on the one hand, carries out molecular marker assisted selection breeding, on the other hand also for other crop powdery mildew gene identification provide reference frame.

Technical scheme

Cardinal principle: first Biology of Plant-Powdery Mildew Interaction gene (MLO) is cloned from barley, research finds that this gene is the special disease-resistant gene of a class, is different from most of NBS (nucleotide-binding site) the type disease-resistant gene of previously clone; Subsequently, investigator has cloned powdery mildew gene in succession from the plants such as tomato, Arabidopis thaliana, pea, capsicum, Root or stem of Littleleaf Indianmulberry, and what research found these genes encodings is all MLO type disease-resistant gene.Subsequently, by test of many times, numerous investigator confirms that MLO type disease-resistant gene has become the distinctive class mildew-resistance gene of plant.Further discovery, plant MLO type gene is a gene family; And Phylogenetic Relationships analysis discovery is carried out to the MLO gene family deriving from different plant species, in different plant species mildew-resistance gene always cluster together, become a class, this class MLO gene all has the characteristic feature of mildew-resistance gene sequence.Completing of bean gene group order-checking provides a convenient approach for excavating powdery mildew gene.Therefore, can by means of the Phylogenetic Relationships of MLO gene family in the Kidney bean full-length genome checked order and the MLO powdery mildew gene of having cloned and for maintaining the conservation of amino acids of powdery mildew gene MLO critical function to identify bean powdery mildew gene.

Key step is as follows:

1) download of Kidney bean whole genome sequence and the collection of MLO type gene thereof

First Kidney bean whole genome sequence is downloaded from Kidney bean sequenced genes group database (http://www.phytozome.net/search.php); Use " DNATOOLS " software to the Kidney bean full-length genome amino acid sequence data building database obtained, then pfam database (protein family database is used, http://pfam.janelia.org/search/sequence) in hidden Markov model (HMM) Blastp (E-value=0.001) sequence alignment is carried out to the aminoacid sequence of MLO structural domain and the Kidney bean full-length genome amino acid sequence database to have set up, preliminary screening goes out candidate gene sequence.Secondly, utilize the MLO type gene order announced, BLAST comparison is carried out to bean gene group database, obtain candidate gene sequence.

2) qualification of Kidney bean MLO type gene family

By the candidate gene of homologous nucleotide sequence obtained in the above results, analyzed by Pfam (E-value=1.O), remove the gene order (Fig. 1) without ' MLO ' structural domain.The ClustalW instrument (Multiple Sequence Alignment program) Candidate Disease Resistant Genes sequence provided by MEGA3.1 software again carries out Multiple Sequence Alignment, removes tumor-necrosis factor glycoproteins.

3) by the Kidney bean MLO type powdery mildew gene of the Phylogenetic Relationships qualification candidate of plant MLO type gene

Because previous research is verified, dicotyledons MLO type powdery mildew gene is positioned at plant MLO Phylogenetic Tree same district group, therefore in Phylogenetic Relationships research, we are MLO type mildew-resistance gene with Kidney bean MLO type gene together with the cluster analysis of the MLO type gene family of Arabidopis thaliana with some other crops, to obtain the Kidney bean mildew-resistance gene (Fig. 2) of candidate.

4) comparison of bean powdery mildew gene and known plant MLO powdery mildew gene

BioXM2.6 software is utilized to convert the aminoacid sequence of the MLO powdery mildew gene of the MLO type powdery mildew gene of Kidney bean candidate and Arabidopis thaliana, tomato, pea, barley the file of Fasta form to, these files are imported BioEdit7.0 software, use Clustal software in this software to carry out Multiple Sequence Alignment, disclose the conservative property in candidate's powdery mildew gene important amino acid residue and region.Thus identify the powdery mildew gene (Fig. 3) of Kidney bean candidate further.

Positively effect of the present invention:

1) shorten the bean powdery mildew gene excavating cycle, be conducive to the Rapid identification of powdery mildew gene.Mildew-resistance gene not only takes time and effort, efficiency is low to adopt ordinary method (map based cloning, transposon tagging etc.) to excavate, and is difficult to successfully.The present invention is based on plant comparative genomics, genetics, bioinformatics method and excavate bean powdery mildew gene fast, not only can shorten the time, powdery mildew gene determination rates can also be improved.

2) Kidney bean (Phaseolus vulgaris) belongs to Papilionaceae Phaseolus, is one of whole world important vegetable of extensively planting.Because Kidney bean hereditary basis is narrow, Germplasm Resources Diversity is low, therefore by conventional molecule marker (RAPD, ISSR, SSR, AFLP etc.) qualification bean powdery mildew genetic comparison difficulty.The candidate's powdery mildew gene exploitation be tested and appraised is divided into from functional indicia (SNP, SCAR etc.) accordingly, can fast for the molecular marker assisted selection of disease-resistant gene, carry out the initiative of multiresistance breeding material, can shortening the breeding cycle, improve breeding efficiency.

3) lay a good foundation for setting forth Kidney bean mildew-resistance molecular mechanism.The qualification of Kidney bean mildew-resistance gene, by transgenic technology, RNAi, virus induced gene silencing (virus induced gene silencing, VIGS) molecular mechanism of the Effect of Anti Powdery Mildew such as technology provides genetic resources, is conducive to the mechanism of action setting forth Kidney bean mildew-resistance fast.

Accompanying drawing explanation

The qualification of Fig. 1 Kidney bean MLO gene;

What this figure showed is 19 MLO type gene identification results, and each gene is containing ' MLO' a conserved domain.

The Phylogenetic Relationships analysis of Fig. 2 plant MLO gene family and the qualification of Kidney bean MLO type powdery mildew gene thereof;

Arabidopis thaliana is the model plant of plant science research, in phylogenetic tree construction, the powdery mildew gene (PsMLO) of 15 MLO type genes (wherein 3 genes are powdery mildew genes: AtMLO02, AtMLO06 and AtMLO12) of Arabidopis thaliana, Gene against Powdery Mildew in Tomato (SlMLO), barley powdery mildew gene (HvMLO and HvMLO02) and pea is selected to and the analysis of Kidney bean MLO type gene clusters.Identify the MLO type powdery mildew gene of 6 Kidney bean candidates altogether.In figure, the gene of red-label is exactly candidate's bean powdery mildew gene.

The compare of analysis of Fig. 3 Kidney bean MLO type powdery mildew gene;

6 bean powdery mildew genes and barley (HvMLO), tomato (SlMLO), pea (PsMLO), Arabidopis thaliana powdery mildew gene (AtMLO02, AtMLO06 and AtMLO12) to compare, qualification powdery mildew infects the conservative type in amino-acid residue and the region played an important role.In figure, TM1-TM7 represents 7 revolving die regions of Kidney bean MLO type powdery mildew gene; Black round dot represents that powdery mildew infects important amino-acid residue; CaMBD represents calmodulin CaM binding region; I and II represents and infects important amino acid region to powdery mildew.

Embodiment

The qualification of disease-resistant gene has important effect in the research of crop disease-resistant theory of heredity and disease-resistant variety seed selection.Present method Rapid identification can go out Kidney bean powdery mildew gene.Specific implementation process is as follows:

1) collection of Kidney bean MLO type gene

In order to obtain the whole MLO type gene family member of Kidney bean, we are first with the MLO type gene of Arabidopis thaliana, the mildew-resistance MLO gene order of tomato, pea, rose, capsicum, Root or stem of Littleleaf Indianmulberry builds HMM model, receives rope MLO type gene from bean gene group sequence, secondly the MLO gene order delivered in Different Crop as target sequence (from DFCI database: TC171015, TC267529, DFCI:TC327983, TC289653, TC312087, TC132500, TC133436, TC317623, TC317025, TC315947, TC325903, TC315944, TC315912, TC322759, TC322059, TC330654, TC282713, TC293173, TC281861, TC283253, TC283383, TC285032, TC290021, TC302716, TC283487, TC282866, TC283441, TC281428, TC285118, TC285090, from GenBank database: AY967408, AF384145, AF384144, AY029312-AY029315, AY029317-AY029319, Z95352, AF369563-AF369565, AF369567, AF369569-AF369576, Z83834, Z95496, AY581255), BLAST comparison is carried out to Kidney bean database (http://www.phytozome.net/search.php), select the highest sequence of similarity to download, obtain the MLO type gene (Phvulv091011469m.g of 19 candidates altogether, Phvulv091024194m.g, Phvulv091024134m.g, Phvulv091023241m.g, Phvulv091008702m.g, Phvulv091000777m.g, Phvulv091000776m.g, Phvulv091019606m.g, Phvulv091019621m.g, Phvulv091019051m.g, Phvulv091001637m.g, Phvulv091001610m.g, Phvulv091018434m.g, Phvulv091009501m.g, Phvulv091009508m.g, Phvulv091010457m.g, Phvulv091024352m.g, Phvulv091024354m.g, Phvulv091030434m.g).

2) qualification of Kidney bean MLO type gene conserved domain

In order to verify these MLO gene accuracys further, we carry out the qualification of conserved domain " MLO " to the MLO gene of these 19 candidates.With the aminoacid sequence of the MLO type gene of each candidate for benchmark, ' the qualification of MLO' conserved domain is carried out in PFAM (http://pfam.sanger.ae.uk/) website, result shows, each MLO type gene has one or more ' MLO ' structural domain (Fig. 1).

3) the Phylogenetic Relationships analysis of Kidney bean MLO type gene

In previous research, find that dicotyledons powdery mildew gene aggregates into district's group; Therefore, in phylogenetic tree construction, we have selected 15 MLO type genes (wherein 3 genes are powdery mildew genes: AtMLO02, AtMLO06 and AtMLO12) of model plant Arabidopis thaliana, the powdery mildew gene of Gene against Powdery Mildew in Tomato, barley powdery mildew gene and pea analyze with Kidney bean MLO type gene clusters together with cluster analysis.Kidney bean MLO type gene and other crop powdery mildew gene protein sequences are carried out multisequencing connection to join (adopting Clustal X1.83 software to carry out), and utilize Genedoc software (http://www.nrbsc.org/gfx/genedoc/index.html) to show multisequencing to join the result of joining.Clustal multisequencing is joined the result of joining output in MEGA4.0 software, and utilize this software to construct adjacent tree (neighbor-joining, NJ) respectively, utilize Bootstrapping method to assess these evolutionary trees.Found that in dicotyledons mildew-resistance gene district group, there is the MLO type powdery mildew gene (see Fig. 2) of 6 Kidney bean candidates.

4) comparison of Kidney bean MLO type disease-resistant gene

In the research of barley MLO type powdery mildew gene, in succession found some important areas and single amino acids in research, they infect large wheat powdery mildew irreplaceable effect.In order to identify in the bean powdery mildew gene of 6 candidates, these important areas and amino acid whether high conservative, we have carried out amino acid alignment analysis to from 3 mildew-resistance genes (AtMLO02, AtMLO6 and AtMLO12) of Arabidopis thaliana, tomato powdery mildew gene (SlMLO), powdery mildew of pea gene (PsMLO).Find the powdery mildew gene of Kidney bean 6 candidates and known MLO type powdery mildew gene 7 cross-film districts, 30 important amino-acid residue, 1 calmodulin CaM binding region (CaMBD) and two regions played an important role with powdery mildew identification (I and II) high conservative (Fig. 3).Show that these six genes are exactly Kidney bean MLO type mildew-resistance gene.

Claims

1. Kidney bean mildew-resistance gene, it is characterized in that being selected from following 6 genes or its one of:

Phvulv091024194m.g

Aminoacid sequence:

MGGRSLEETPTWAVAVVCFVLLTISIVIDHIFHLIGKWLKKKHKRALYESLEKIKSELMLLGFISLLLTVGQSVISRICISEKVAGTFHPCTHKTDHTRRLLPELFRSDHNQNLRRILAAGGTDKCAAQGKVPFVSSEGIHQLHIFIFVLAVFHVLYCILTLALGRAKMRRWKRWEEETKTAQYQFSHDPERFRFARETSFGRRHLSFWTQHTILVWIICFFRQFVRSVPKVDYLTLRHGFMMAHLGPQSRHKFDFRQYIKRSLEEDFKVVVEISPPIWFITVLFLLFHTHGWYSYLWLPFAPLIIVLLVGTKLQVIITKMGQRIQERGEVVKGMPLVQPGDDLFWFNKPRLILYLVNFVLFQNAFQLAYFSWTAVQFGMKSCFHSQTEDVVIKISMGVFVQFLCSYVTLPLYALVTQMGSTMKPTIFNERVAMALRNWHHAAKKHVKQQNRRLQSRPTTPNHSTSQAHLLRRCHSEIDIYPTDSEAHHPYEMDLSPSSKVHDHEVEMEMGHLDHGPLEDVNELNSALAGSGVTQHEINIEHGKEFSFDNR

Nucleotide sequence:

ATGGGAGGAAGAAGCTTGGAGGAAACTCCTACTTGGGCTGTAGCCGTTGTTTGCTTCGTTTTGCTCACCATATCTATCGTCATCGACCACATCTTCCATCTCATAGGAAAGTGGTTGAAGAAGAAGCACAAAAGAGCACTATATGAGTCACTTGAAAAGATCAAATCAGAACTTATGTTATTGGGGTTCATATCGCTGCTTCTAACGGTAGGACAAAGTGTAATATCGAGGATATGTATATCAGAAAAGGTTGCAGGGACATTCCACCCCTGCACTCACAAAACAGACCACACCCGCAGATTACTACCGGAGCTTTTCCGTTCCGATCACAACCAAAATCTACGCCGTATTTTGGCGGCGGGAGGAACTGACAAGTGTGCGGCACAGGGTAAAGTCCCGTTTGTCTCATCAGAGGGCATTCATCAACTCCATATATTTATCTTCGTGCTGGCTGTTTTTCACGTTCTTTACTGCATTCTCACTCTGGCTCTTGGTAGAGCAAAGATGAGAAGGTGGAAACGGTGGGAAGAGGAAACAAAAACAGCACAGTATCAATTTTCACACGATCCTGAACGGTTTAGATTTGCGAGGGAGACTTCGTTTGGAAGAAGACACTTGAGTTTCTGGACTCAACACACTATCCTAGTTTGGATTATTTGTTTCTTCAGGCAGTTTGTACGCTCAGTTCCTAAAGTTGATTACTTGACCTTGAGGCATGGATTTATGATGGCGCATTTGGGACCTCAAAGTCGCCATAAATTCGACTTTAGGCAATATATCAAAAGGTCTTTGGAAGAGGACTTCAAAGTGGTCGTTGAAATCAGTCCTCCAATCTGGTTCATCACAGTGCTTTTTCTCCTGTTTCACACCCATGGCTGGTACTCTTATCTGTGGCTGCCATTTGCTCCTTTGATAATTGTTCTATTAGTGGGAACGAAGCTGCAAGTGATAATAACGAAGATGGGTCAGAGAATTCAAGAAAGAGGAGAGGTTGTAAAGGGCATGCCACTGGTGCAGCCTGGGGATGATCTCTTCTGGTTTAACAAACCTCGACTTATTCTCTACCTCGTCAATTTCGTGCTCTTTCAGAACGCTTTCCAACTTGCCTACTTTTCATGGACTGCGGTTCAGTTTGGGATGAAATCCTGTTTTCATTCGCAGACGGAGGATGTGGTGATTAAAATCTCAATGGGGGTTTTCGTTCAATTCCTTTGCAGCTACGTAACTCTTCCTCTCTATGCTCTCGTCACACAGATGGGTTCAACCATGAAACCAACCATATTCAACGAAAGAGTAGCCATGGCTCTGAGGAATTGGCACCATGCAGCCAAGAAACACGTCAAGCAACAGAACCGTAGATTACAGTCAAGGCCCACAACCCCCAATCACTCTACATCTCAGGCCCATCTCTTACGCCGCTGCCACAGTGAAATTGACATCTATCCCACTGACTCTGAGGCCCATCATCCCTATGAGATGGATTTGAGCCCATCATCCAAGGTGCATGATCACGAAGTGGAAATGGAAATGGGCCACTTGGATCATGGCCCACTAGAAGATGTGAATGAGTTGAACTCAGCCCTTGCGGGATCAGGTGTGACTCAACACGAGATTAATATTGAACACGGCAAGGAGTTTTCATTTGATAATAGATGA

Phvulv091024134m.g

Aminoacid sequence:

MGGNGGRNLDETPTWAVAVVCFVLLSVSIIIEYIFNLIGKWLKQKHKRALYESLEKIKSELMLLGFISLLLTVGQGLISRICISEKVAGTFHPCAHRRVKQNNTPPLEHDEYRNNRHLLALEDKCAPQGKVPFVSSEAIHQLHIFIFVLAVFHVLYCILTHALGRAKMRRWKRWEVETKTPEYQVSHDPQRFRFARETSFGRRHLSFWTQNAVLVWIICFFRQFVHSVPKVDYLTLRHGFMMAHLGPHSHQKFDFGKYIKRSLEEDFKVVVEISPPIWFITVLFLLFNTHGWYSYLWLPFAPLIIVLLVGTKLQVIITKMGQRIQERGEVVKGMPVVQPGDHLFWFNKPRLILYLINFVLFQNAFQLAFFSWAALQFMMKSCFQSQKQDVVIRISMGIFVQFLCSYVTLPLYALVTQMGSTMKATIFNEKVAMALRNWHHTAKKNVKEKRGVRSQSPLSTRPSTPQQQPKSQANLLRRYHSEMATYPSSPIRFDFEAHLPYGIHSPPSSMVNAAATSSIHRHEMEMETEIEMDQVSALTQRQIDIDIDIQDNQEFSFAKR

Nucleotide sequence:

ATGGGAGGAAATGGAGGAAGGAATTTAGACGAAACACCAACCTGGGCTGTGGCCGTTGTTTGCTTTGTTTTGCTCTCTGTATCTATCATCATCGAATACATCTTCAATCTAATAGGAAAGTGGTTGAAGCAGAAACACAAAAGAGCTCTGTACGAGTCACTTGAAAAGATCAAATCAGAGCTCATGTTATTAGGGTTCATATCGTTGCTCCTAACGGTAGGGCAAGGTCTAATATCGAGGATATGCATATCAGAAAAGGTTGCAGGGACATTTCACCCTTGCGCTCATAGAAGAGTGAAGCAAAACAACACTCCCCCATTAGAACACGATGAATACCGAAATAACCGCCATCTTTTAGCGTTGGAAGACAAGTGTGCACCGCAGGGTAAAGTGCCATTTGTGTCATCGGAAGCCATTCACCAGCTCCATATATTTATCTTCGTGCTGGCTGTTTTTCACGTGCTTTACTGCATTCTCACTCACGCCCTTGGTAGAGCAAAGATGAGAAGGTGGAAGCGATGGGAAGTAGAAACCAAAACACCAGAGTACCAAGTTTCACACGATCCTCAACGATTCAGATTTGCAAGAGAGACATCGTTTGGAAGAAGACACCTAAGTTTCTGGACCCAGAATGCTGTCCTGGTTTGGATTATTTGTTTCTTCAGACAGTTTGTACACTCAGTTCCTAAAGTTGATTACTTGACCTTGAGACATGGATTTATGATGGCACATTTGGGGCCTCACAGTCACCAGAAATTCGACTTTGGGAAGTACATCAAAAGATCTTTGGAAGAGGACTTCAAAGTGGTCGTTGAAATCAGTCCTCCAATCTGGTTCATCACAGTGCTTTTTCTCCTATTTAATACTCACGGCTGGTACTCTTATCTGTGGCTGCCATTTGCTCCTTTGATTATTGTTCTATTAGTGGGAACGAAGCTGCAAGTGATTATAACGAAGATGGGTCAGAGAATTCAAGAACGAGGAGAGGTTGTAAAGGGCATGCCAGTGGTGCAACCTGGGGATCACCTCTTCTGGTTTAACAAACCTCGACTTATTCTCTACCTCATTAATTTTGTGCTCTTTCAGAATGCTTTTCAGCTTGCTTTCTTTTCATGGGCTGCGCTTCAATTTATGATGAAGTCATGTTTCCAGTCCCAAAAGCAGGATGTGGTGATCAGAATCTCAATGGGGATCTTCGTTCAATTCCTTTGCAGCTACGTCACTCTTCCTCTCTATGCTCTCGTGACGCAGATGGGTTCAACAATGAAGGCAACGATTTTCAACGAAAAAGTAGCGATGGCTCTAAGAAATTGGCACCACACTGCGAAGAAGAACGTGAAAGAGAAGCGTGGAGTACGATCGCAGAGTCCTTTGTCAACTCGGCCTTCAACCCCGCAGCAGCAGCCAAAGTCGCAGGCTAATCTGCTGAGACGGTACCACAGTGAAATGGCGACTTATCCATCTTCTCCGATAAGGTTCGATTTTGAGGCACATCTTCCTTATGGGATCCATTCACCCCCCTCCTCCATGGTTAATGCTGCAGCTACTTCTTCTATTCATCGCCATGAAATGGAGATGGAAACGGAAATTGAAATGGATCAGGTCTCAGCTCTCACTCAACGCCAGATTGATATTGACATTGACATTCAAGACAATCAAGAATTTTCTTTTGCTAAAAGATAA

Phvulv091009058m.g

Aminoacid sequence:

MAGGGGRNLEETPTWAVSTVCFVLIFISIIIEHIIHLIGQWLKKKRKRALYESLEKIKSELMLLGFLSLLLTVGQGLVSRICISEKIASTWHPCSADDTHSEESGQGTNSRRLLAAFHGSNDVNPRRVLAGGGSDKCGEGKVPFVSSEGIHQLHIFIFVLTVFHVLYCILTMALGRAKMKRWKRWEEETKTPEYQFSHDPERFRFARETSFGRRHLSFWTKNPVLMWIVCFFRQFVRSVPKVDYLTLRHGFIMAHLAPQSHSKFDFRKYIKRSLDEDFKVVVGISPPFWLFAVLFLLLNTHGSYSYLWLPFIPLIIILLVGTKLQVIITEMGLRIQQRGEILKGVPLVQPGDYLFWFNKPGLILYLINFVLFQNAFQLAFFSWSVLQFGIKSCFHAHTEDVVIRITMGVLlQILCSYVTLPLYALVTQMGSTMKPTIFNERVAEALRNWHQTAKKQIRQNRVGPLSLSVTPMSSRPTTPSHNMSPVHLLRYYRSEIDSFPTSPRRSNVDGDHTQPWDVESPSPSYSHHEMEMGHTNDPTTNTTHHEIVPAANTREFSFDKRPTTAP

Nucleotide sequence:

ATGGCAGGTGGCGGAGGAAGAAACTTGGAGGAAACACCCACATGGGCCGTCTCCACTGTCTGTTTTGTTCTGATCTTTATATCTATAATCATCGAACACATCATCCACCTCATAGGACAGTGGTTGAAGAAGAAGCGCAAAAGAGCGCTGTACGAGTCACTCGAAAAGATCAAATCAGAGCTTATGTTATTGGGGTTCTTGTCGTTGCTTTTAACGGTAGGACAAGGTCTGGTATCAAGAATATGCATATCAGAAAAAATTGCATCGACATGGCACCCTTGTAGCGCCGACGACACGCATAGTGAAGAATCAGGACAGGGAACCAACAGCCGCAGGTTGTTAGCGGCGTTCCATGGTTCAAACGATGTAAATCCACGCCGTGTTTTGGCGGGAGGAGGAAGTGACAAGTGTGGCGAGGGTAAAGTCCCGTTTGTGTCATCGGAGGGAATCCATCAACTCCATATATTTATCTTTGTGCTAACTGTTTTTCATGTCCTTTACTGCATTCTCACCATGGCTCTGGGAAGAGCAAAGATGAAGAGGTGGAAACGATGGGAAGAGGAAACCAAGACACCAGAGTACCAATTTTCACACGATCCTGAACGATTTAGATTTGCGAGAGAAACATCGTTTGGAAGAAGGCATTTGAGTTTCTGGACCAAAAACCCTGTTCTCATGTGGATTGTTTGTTTCTTTAGGCAATTTGTGCGATCAGTTCCTAAAGTGGATTACTTGACCTTAAGACATGGATTTATCATGGCACATTTGGCTCCTCAAAGTCACTCCAAATTTGATTTTCGGAAATACATAAAACGATCACTGGATGAGGATTTCAAAGTCGTTGTTGGAATCAGTCCTCCATTCTGGTTGTTCGCGGTGCTGTTTCTCCTCTTGAACACTCATGGCTCGTACTCTTATCTGTGGCTGCCATTCATTCCTTTGATTATCATTCTGTTAGTTGGAACCAAGTTGCAAGTGATCATAACTGAGATGGGTCTAAGAATTCAACAAAGAGGAGAGATTCTGAAGGGAGTGCCACTGGTTCAACCAGGAGATTATTTGTTCTGGTTTAACAAACCTGGTCTTATTCTTTACCTTATTAACTTTGTGCTCTTTCAGAATGCTTTTCAGCTTGCTTTCTTCTCCTGGTCTGTGCTTCAATTTGGGATTAAATCTTGTTTCCACGCACATACAGAGGATGTAGTGATCAGAATCACAATGGGGGTTCTTATTCAAATCCTGTGCAGCTACGTTACTCTTCCTCTCTATGCCCTAGTTACACAGATGGGTTCAACAATGAAGCCAACGATATTCAACGAAAGAGTTGCTGAGGCACTGCGGAACTGGCACCAGACCGCGAAGAAGCAGATAAGACAGAACCGTGTGGGACCCTTGTCGTTGTCGGTGACACCGATGTCGAGCAGACCCACAACCCCGAGCCACAACATGTCCCCAGTGCACCTCTTGCGCTACTACCGTTCTGAAATTGACAGCTTCCCCACCTCTCCACGACGTTCGAACGTCGACGGCGACCACACCCAGCCCTGGGACGTTGAATCCCCTTCTCCTTCCTACTCGCACCACGAAATGGAAATGGGTCACACCAATGACCCCACCACTAACACAACCCACCATGAGATTGTTCCTGCTGCAAACACCAGGGAATTCTCCTTTGATAAAAGACCTACCACAGCTCCGTAG

PhVulv091023241m.g

Aminoacid sequence:

MGKEEVLQRKLEATPTWVVAAVCFVMLAISIIIEHVIEELGKWLKKKHKKALHEALEKIKGELMLLGFLSFLLVVFEDRIVSICIPESVASTWNPCDPSYVSKGAEGYVEKCAKKKEGTVAFMSKYGLHQLHIFVFVLAIFHILQCIMTLTLGRTKISIWRKWEDETKGLEHQFSHDPERFRFARDTTFGRRHLNSWSKSPTLLWIVSFCRQFYGSLNKADYMALRHGFVVAHLTPASEPKFDFQNYITRTLDEDFAVVVGISPTIWFFAVVILLTNTHGWYSYFWVPFIPLIIILLVGTKLQMIITDMALKIQDRGEVVKGEPVVQPGDELFWFNNPRLVLFLIHLVLFLNAFQLAFFTWSTLDNGFKINSCFHRTTADIVIRLTMGVLTQVLCSYVTLPLYALVIQMGSSMKPTIFKDNVASALKSWHHSAKKHLKHNRDSTSNTPFSSRPGTPTRGMSPVHLLHKHPRHSDSPLVSPRASDYENGQWDAEVSHSPSHHARDIDHEETMQVQPPAAAELPPAGLNPVRTQHEINIALSEFSFGRGRNPGRADEY

Nucleotide sequence:

ATGGGCAAAGAAGAAGTTCTACAGAGGAAGTTGGAGGCAACTCCTCATGGGTAGTTGCAGCTGTGTGCTTTGTGATGCTTGCTATTTCAATCATCATTGAACATGTTATTGAAGAACTTGGAAAGTGGTTAAAAAAGAAACACAAAAAGGCTCTTCATGAAGCGCTGGAAAAGATCAAAGGAGAGCTTATGCTGCTGGGATTCCTATCCTTTCTCTTAGTGGTGTTTGAAGATCGAATTGTTTCTATCTGCATCCCAGAAAGTGTTGCATCCACTTGGAATCCTTGTGATCCCAGTTATGTGAGTAAAGGTGCAGAAGGATATGTTGAGAAATGCGCAAAAAAAAAGGAAGGCACAGTTGCTTTTATGTCTAAATATGGGCTTCACCAGCTCCATATATTCGTCTTTGTGCTTGCTATTTTTCACATTCTACAGTGCATCATGACACTGACTTTGGGTAGAACTAAGATATCAATATGGAGGAAGTGGGAAGACGAAACAAAGGGTCTTGAACATCAGTTCTCCCATGATCCTGAGAGGTTCAGGTTTGCTAGGGATACAACGTTTGGACGAAGGCACTTGAACTCATGGAGTAAATCACCAACTTTATTATGGATAGTTAGCTTCTGCAGACAATTCTATGGATCACTTAATAAAGCTGACTATATGGCATTACGGCATGGATTCGTCGTGGCACATTTGACTCCAGCAAGTGAGCCAAAATTTGACTTCCAGAACTATATCACCAGAACACTAGATGAGGATTTTGCAGTAGTGGTGGGCATAAGTCCAACCATATGGTTCTTTGCAGTGGTAATTCTGCTCACAAATACTCACGGGTGGTATTCTTATTTTTGGGTTCCATTTATCCCACTAATTATAATCTTGTTGGTGGGAACAAAGCTACAAATGATCATAACAGATATGGCACTAAAGATTCAAGATAGGGGGGAAGTGGTCAAGGGTGAACCAGTGGTTCAGCCAGGAGATGAATTGTTCTGGTTCAATAATCCTCGCCTCGTTCTCTTTCTGATTCATCTTGTTCTGTTCCTGAATGCATTTCAATTGGCGTTTTTTACTTGGAGCACATTAGACAATGGGTTCAAAATAAACTCCTGCTTTCACCGAACTACTGCAGATATTGTCATTAGACTTACAATGGGGGTTCTCACCCAAGTTCTATGCAGCTATGTGACTTTGCCTCTTTATGCTCTAGTCATACAGATGGGTTCTAGCATGAAACCCACCATTTTCAAAGATAACGTGGCATCAGCACTAAAGAGCTGGCATCACTCTGCTAAAAAGCATCTGAAACACAACAGGGATTCAACTTCTAACACACCATTCTCAAGCAGACCAGGAACCCCAACTCGTGGCATGTCTCCAGTGCACCTGCTTCACAAGCACCCTAGACACAGTGATAGTCCACTAGTTTCTCCAAGAGCATCCGATTATGAAAATGGGCAATGGGATGCTGAAGTGTCACATTCCCCAAGCCACCATGCAAGAGATATTGATCATGAAGAGACCATGCAGGTGCAGCCTCCAGCAGCAGCAGAATTGCCTCCTGCTGGACTCAATCCTGTTCGAACTCAACATGAAATCAACATTGCTTTATCTGAATTTTCATTTGGGAGGGGACGCAACCCTGGTAGAGCAGATGAGTATTAG

Phvulv091000777m.g

Aminoacid sequence:

MANEVYERTLEETPTWAVAVVCFLLLAVSIAIEHLIHGIGKWFKKKQKNALFEALEKVKGELMVLGFISLLLSVLQDQISKICVSEEVAASWHPCANPKSSTATKTDEDESDDIQINSRKLLQLYDSIPRRILATKGYDKCAEKGKVAFVSAYGIHQLHIFIFVLAIFHILQCIITLGLGRTKMRKWRAWENETKTIEYQFYNDPERFRFARDTTFGRRHLNPWSQSTISLWIVSFFRQFFGSATKVDYLTLRHGFITAHLAPGSDARFDFQKYIQRSLEKDFKVVVGISPVIWFFAVLFLLTNTHGWYSSYWLPFIPFVVILLVGAKLQMIITKMGLRITDRGEVVKGAPLVEPGDDLFWFNRPRLLLFLIHLVLFQNAFQLAFFSWSTYEFSLDSCFHETTADVAIRLTMGVVIQVLCSYVTLPLYALVTQMGSTMKPTIFNQRVASALKNWHSTAKKQVKNSKQNTPFSSKPSTPTYGMSPMHLLQKHLAGRSDSAQTSPRTSNYENEQWDVEGSPSTSNHAAAEETQMQVMEPGSSSAPEFPTSSQIEIRVSSSEFSFEKRHTGASEEI

Nucleotide sequence:

ATGGCAAACGAAGTTTATGAGCGAACTCTGGAGGAAACACCAACATGGGCTGTTGCAGTTGTGTGTTTTTTGCTTCTTGCTGTTTCAATCGCCATAGAACATCTTATTCATGGTATTGGAAAGTGGTTCAAGAAGAAACAAAAAAATGCTCTTTTCGAAGCGTTGGAGAAGGTCAAAGGAGAGCTTATGGTGCTAGGATTCATATCCTTGCTCCTAAGTGTGTTGCAAGATCAAATTTCCAAGATCTGCGTATCAGAAGAAGTTGCAGCTTCGTGGCATCCCTGTGCCAACCCAAAGTCCTCAACTGCAACAAAAACTGATGAGGATGAATCTGATGACATTCAGATAAATTCTAGGAAACTCCTACAACTTTATGACTCCATACCAAGGCGTATTCTGGCTACAAAAGGTTACGACAAGTGTGCTGAAAAGGGAAAAGTAGCTTTTGTTTCAGCCTATGGGATTCATCAGCTGCATATATTCATCTTTGTGTTAGCAATATTTCACATCCTACAGTGCATTATAACACTGGGTTTGGGAAGAACCAAGATGAGAAAGTGGAGGGCATGGGAAAACGAAACCAAGACCATTGAATATCAATTTTATAACGATCCGGAGAGGTTCAGGTTTGCAAGGGACACAACATTTGGAAGAAGACACTTGAATCCATGGAGTCAGTCAACAATTTCGTTATGGATAGTTAGTTTCTTCCGACAATTCTTTGGATCAGCCACCAAAGTCGACTATTTGACATTACGACATGGATTCATCACGGCGCATCTGGCACCGGGAAGTGATGCAAGATTTGATTTCCAAAAGTACATCCAAAGATCACTGGAGAAGGATTTTAAAGTTGTTGTGGGAATAAGCCCAGTGATATGGTTCTTTGCTGTGCTATTCCTTCTCACAAATACTCATGGGTGGTATTCTTCTTATTGGCTTCCATTCATCCCATTTGTTGTAATCTTATTAGTGGGTGCTAAACTTCAAATGATCATAACAAAGATGGGACTGAGGATTACAGACAGAGGGGAAGTTGTCAAAGGTGCACCTCTGGTTGAGCCAGGAGATGACCTGTTCTGGTTCAATCGCCCACGTCTCCTCCTCTTTCTCATTCATCTTGTGCTTTTTCAGAATGCTTTTCAACTGGCATTTTTCTCTTGGAGTACTTATGAATTCTCCCTAGACTCTTGCTTCCATGAAACAACTGCAGATGTTGCCATAAGACTTACAATGGGGGTTGTCATACAAGTTCTATGCAGCTATGTGACATTACCTCTATATGCTCTAGTCACACAGATGGGTTCAACCATGAAGCCTACCATCTTCAATCAGAGAGTGGCATCAGCACTGAAGAACTGGCACAGCACTGCCAAAAAGCAAGTCAAAAACAGCAAGCAAAACACACCATTCTCAAGCAAGCCATCAACCCCAACATATGGTATGTCTCCAATGCATCTGTTGCAAAAACACCTTGCTGGTAGAAGTGACAGTGCACAAACATCTCCAAGGACATCAAACTATGAAAATGAACAATGGGATGTTGAAGGCTCACCTTCCACAAGCAACCATGCAGCAGCTGAAGAAACTCAAATGCAAGTCATGGAACCAGGCTCAAGCTCAGCACCAGAATTTCCTACCAGCTCTCAGATTGAAATCAGAGTTAGTTCATCAGAGTTTTCCTTTGAGAAGAGACACACTGGTGCTAGTGAGGAGATATGA

Phvulv091000776m.g

Aminoacid sequence:

MGEAKIHKRSLEETPTWAFAVVCFMLLAISIIIELVIHAIGKWLRKKHKSGLYESLEKVKGELMMLGFLSMLLTVFQDPLSKICISQNAASTWHPCSNPKAVSNSNATSHTIDRKLLHLDPIPRRVLAAKGYDKCADKGKVAFVSAYGIHQLHIFIFVLAIFHIIQCIVTLALGRTKMRRWKKWEDETKTIEYQFYNDSKRFRLAKDTTFGQRHLNTWSQSSISLWIVSFFRQFFGSVKKVDYFVLRHGFIMAHVSPRSDSGFDFQRYIKRSLDEDFKVVVGISPIIWFEAVLFLLTNTHGWYSNYWLPFIPLITILIVGADLQMIITKMGLRIQDRGVVMGAPVVEPGDDLFWFNRPRLLLFIIHLVLFQNAFQLAHFAWSTYEFSIESCFHKTNAENVVRLTMGVVTQVLCSYVTLPLYALVTQMGSTLKPTIFNDRMAAALKKWHHTSKKHLKHSQHSEAKKSIPFSSTSSSPIFGMSPIHLLHRHPVGRSDSAQTSPRTSLYENQQCDVQGESSVSNHPGTNETQMQVLGSPQTT

Nucleotide sequence:

ATGGGTGAAGCCAAAATCCATAAAAGATCACTGGAGGAAACACCAACCTGGGCGTTTGCAGTTGTTTGCTTTATGCTGCTTGCTATTTCAATCATCATTGAGCTTGTTATTCATGCTATTGGAAAGTGGTTAAGAAAGAAACACAAAAGTGGTCTTTATGAGTCGTTGGAAAAGGTCAAAGGAGAGCTTATGATGCTAGGGTTCCTATCTATGCTCCTAACTGTGTTCCAAGATCCACTTTCTAAGATCTGCATATCACAAAATGCTGCATCAACATGGCATCCTTGCTCCAACCCAAAGGCCGTGAGTAATTCTAACGCAACATCTCATACCATTGATAGGAAACTTCTCCATTTGGACCCCATTCCACGACGTGTTCTAGCTGCAAAAGGTTATGACAAATGTGCAGACAAGGGAAAAGTTGCTTTTGTTTCAGCATATGGGATTCACCAGCTCCATATATTCATCTTTGTGTTAGCAATTTTTCATATCATACAATGCATTGTAACACTAGCTTTGGGAAGAACTAAGATGAGACGCTGGAAGAAGTGGGAAGACGAAACAAAGACAATTGAATACCAATTCTATAATGATTCTAAGAGGTTCAGGCTTGCAAAGGACACAACATTTGGACAAAGGCACTTGAATACTTGGAGTCAGTCATCGATTTCCTTATGGATAGTTAGCTTCTTCAGACAATTCTTTGGATCGGTTAAGAAAGTCGACTATTTTGTTTTACGACATGGATTTATCATGGCACATGTGTCACCGAGAAGTGATTCAGGATTTGATTTCCAGAGGTATATCAAAAGATCACTTGACGAGGATTTTAAAGTTGTGGTAGGCATAAGCCCAATTATTTGGTTCTTTGCGGTTCTCTTCCTGCTGACTAATACTCATGGGTGGTATTCTAACTATTGGCTTCCATTCATCCCATTAATTACAATTTTGATAGTGGGTGCTGATCTACAAATGATCATAACAAAGATGGGACTAAGGATTCAAGACAGAGGAGTAGTTATGGGTGCACCTGTGGTTGAGCCTGGAGATGACTTGTTTTGGTTCAACCGTCCACGACTCCTTCTCTTCATCATTCATCTTGTTCTTTTTCAGAATGCCTTTCAATTAGCACATTTTGCATGGAGTACATATGAATTCTCCATAGAATCTTGCTTCCACAAAACAAATGCAGAGAATGTGGTTAGACTTACAATGGGGGTTGTAACACAAGTTCTATGCAGCTATGTGACTTTGCCTCTTTATGCTCTAGTTACACAGATGGGTTCAACCTTGAAACCAACCATTTTCAATGATAGAGTAGCAGCAGCATTGAAGAAATGGCACCATACTTCCAAAAAACATCTCAAACACAGCCAACACTCTGAGGCTAAAAAATCAATACCATTCTCCAGCACATCATCAAGCCCAATATTTGGCATGTCTCCCATTCACCTGCTGCATAGACACCCTGTTGGAAGAAGTGACAGTGCACAAACTTCTCCAAGGACCTCCCTCTATGAAAATCAACAATGTGATGTGCAAGGTGAATCTTCCGTAAGCAACCATCCAGGAACAAATGAGACCCAAATGCAAGTTTTGGGTTCTCCTCAAACAACATAA。

2. the application of Rapid identification bean powdery mildew gene described in claim 1, comprising:

1) initiative or the new variety initiative of the breeding material of mildew-resistance MLO gene is carried: utilize the MLO GENE SOURCES given by right 1 to be parent material, in hybridization, backcross progeny, the breeding material that transformation has MLO resistant gene can be obtained.

2) mildew-resistance fundamental research: molecular marker analysis is carried out to the MLO type mildew-resistance gene of right 1; Molecular Mapping or the assignment of genes gene mapping are carried out to this gene; This gene is cloned and Interaction among genes analysis.

3) mildew-resistance transgenic research: utilize the MLO type powdery mildew gene given by claim 1 to carry out transgenic research.