CN116411112A - 一种检测小麦白粉菌抗三唑类杀菌剂的分子标记方法 - Google Patents
一种检测小麦白粉菌抗三唑类杀菌剂的分子标记方法 Download PDFInfo
- Publication number
- CN116411112A CN116411112A CN202211404362.8A CN202211404362A CN116411112A CN 116411112 A CN116411112 A CN 116411112A CN 202211404362 A CN202211404362 A CN 202211404362A CN 116411112 A CN116411112 A CN 116411112A
- Authority
- CN
- China
- Prior art keywords
- powdery mildew
- wheat powdery
- cyp51
- detecting
- primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000209140 Triticum Species 0.000 title claims abstract description 42
- 235000021307 Triticum Nutrition 0.000 title claims abstract description 42
- 241000221785 Erysiphales Species 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 14
- 230000000855 fungicidal effect Effects 0.000 title claims abstract description 8
- 239000000417 fungicide Substances 0.000 title claims abstract description 8
- 150000003852 triazoles Chemical class 0.000 title claims abstract description 8
- 238000001514 detection method Methods 0.000 claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 13
- 238000000137 annealing Methods 0.000 claims description 9
- 239000003147 molecular marker Substances 0.000 claims description 9
- 101710146773 Lanosterol 14-alpha demethylase Proteins 0.000 claims description 7
- 238000012408 PCR amplification Methods 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 238000004925 denaturation Methods 0.000 claims description 6
- 230000036425 denaturation Effects 0.000 claims description 6
- 239000003550 marker Substances 0.000 claims description 5
- 238000003205 genotyping method Methods 0.000 claims description 3
- 238000012257 pre-denaturation Methods 0.000 claims description 3
- WURBVZBTWMNKQT-UHFFFAOYSA-N 1-(4-chlorophenoxy)-3,3-dimethyl-1-(1,2,4-triazol-1-yl)butan-2-one Chemical compound C1=NC=NN1C(C(=O)C(C)(C)C)OC1=CC=C(Cl)C=C1 WURBVZBTWMNKQT-UHFFFAOYSA-N 0.000 claims 1
- 230000035772 mutation Effects 0.000 abstract description 18
- 206010059866 Drug resistance Diseases 0.000 abstract description 10
- 239000003814 drug Substances 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 2
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 2
- 230000003321 amplification Effects 0.000 abstract 1
- 150000001413 amino acids Chemical class 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 230000000844 anti-bacterial effect Effects 0.000 description 6
- 239000003899 bactericide agent Substances 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 5
- 102100021695 Lanosterol 14-alpha demethylase Human genes 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 238000012864 cross contamination Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 235000013339 cereals Nutrition 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 240000005979 Hordeum vulgare Species 0.000 description 2
- 235000007340 Hordeum vulgare Nutrition 0.000 description 2
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 235000009754 Vitis X bourquina Nutrition 0.000 description 2
- 235000012333 Vitis X labruscana Nutrition 0.000 description 2
- 240000006365 Vitis vinifera Species 0.000 description 2
- 235000014787 Vitis vinifera Nutrition 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 244000000004 fungal plant pathogen Species 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000007480 sanger sequencing Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- FFSJPOPLSWBGQY-UHFFFAOYSA-N triazol-4-one Chemical compound O=C1C=NN=N1 FFSJPOPLSWBGQY-UHFFFAOYSA-N 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- PXMNMQRDXWABCY-UHFFFAOYSA-N 1-(4-chlorophenyl)-4,4-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)pentan-3-ol Chemical compound C1=NC=NN1CC(O)(C(C)(C)C)CCC1=CC=C(Cl)C=C1 PXMNMQRDXWABCY-UHFFFAOYSA-N 0.000 description 1
- HYZJCKYKOHLVJF-UHFFFAOYSA-N 1H-benzimidazole Chemical compound C1=CC=C2NC=NC2=C1 HYZJCKYKOHLVJF-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 241001480061 Blumeria graminis Species 0.000 description 1
- 101150065528 CYP51 gene Proteins 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000221787 Erysiphe Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 101000615488 Homo sapiens Methyl-CpG-binding domain protein 2 Proteins 0.000 description 1
- 241001299819 Hordeum vulgare subsp. spontaneum Species 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 102100021299 Methyl-CpG-binding domain protein 2 Human genes 0.000 description 1
- 239000005822 Propiconazole Substances 0.000 description 1
- 229920001131 Pulp (paper) Polymers 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 239000005839 Tebuconazole Substances 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 244000000005 bacterial plant pathogen Species 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000007847 digital PCR Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000008686 ergosterol biosynthesis Effects 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- STJLVHWMYQXCPB-UHFFFAOYSA-N propiconazole Chemical compound O1C(CCC)COC1(C=1C(=CC(Cl)=CC=1)Cl)CN1N=CN=C1 STJLVHWMYQXCPB-UHFFFAOYSA-N 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000008261 resistance mechanism Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 102220084572 rs863225027 Human genes 0.000 description 1
- 108700004121 sarkosyl Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229940045885 sodium lauroyl sarcosinate Drugs 0.000 description 1
- KSAVQLQVUXSOCR-UHFFFAOYSA-M sodium lauroyl sarcosinate Chemical compound [Na+].CCCCCCCCCCCC(=O)N(C)CC([O-])=O KSAVQLQVUXSOCR-UHFFFAOYSA-M 0.000 description 1
- ZNVGYHOBTCWGTO-UHFFFAOYSA-N solutin Natural products Cc1cc(O)cc2OC(C)(O)C(=O)c12 ZNVGYHOBTCWGTO-UHFFFAOYSA-N 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Botany (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明提供了一种检测小麦白粉菌抗三唑类杀菌剂的分子标记方法，属于杀菌剂抗性分子检测领域。利用引物扩增小麦白粉菌抗药性基因cyp51，区分抗性基因位点是否发生突变，该方法可精确区分出所含基因在CYP51‑458位点存在的三种突变类型：突变型（T)、野生型（A),还是杂合型（A/T），提高了对抗性基因cyp51的选择效率，精准预测田间抗药性的频率，科学指导生产用药。
Description
技术领域
本发明属于植物病原真菌抗药性分子检测技术领域，具体涉及一种与小麦白粉菌抗药性相关的KASP标记及其应用。
背景技术
由专性寄生菌（Blumeriagraminisf.sp.tritici, Bgt）引起的小麦白粉病，是小麦生产中最重要病害之一。该病害在小麦整个生育期均可发生，尤其影响籽粒灌浆，造成空瘪粒增加，导致产量下降，我国常年发生面积 685-730万公顷，一般发病田会减产5%～10%，重病田减产达 20%以上。化学药剂为当前防治小麦白粉病的重要防治措施，在保障小麦安全生产中发挥着重要作用。目前，生产上防治小麦白粉病药剂主要以靶向14-α脱甲基酶的麦角甾醇合成抑制剂，唑类药剂为主。国内登记防治小麦白粉病杀菌剂中，三唑类已占到89.9%（单剂和复配），主要以三唑酮、戊唑醇、丙环唑等药剂为主，但大面积、长时间使用作用机制相同的杀菌剂会给病原菌群体造成选择压力，逐渐形成抗药性菌群，最终导致药剂防效丧失。
CYP51 氨基酸突变是植物病原真菌中报道最为广泛的抗性机制，其原因为：CYP51基因在杀菌剂的长期选择下发生单个或多个碱基突变，导致氨基酸序列发生替换，从而引起 CYP51 的结构与空间构象发生改变，使得 CYP51与杀菌剂的结合能力下降，真菌对药剂的敏感性降低导致抗药性产生。在植物病原菌中，CYP51氨基酸广泛存在的突变类型为Y136F，即其136 位酪氨酸被取代为了苯丙氨酸，该突变位点在许多病原菌的抗性菌株中均有报道，如小麦白粉病、葡萄白粉病菌Erysiphenecator、香蕉黑条叶斑病菌Mycosphaerellafijiensis和小麦叶锈病菌Pucciniatriticina。因此，CYP51 氨基酸突变可用于分子监测病原体群体的抗药性，从而预测该病原菌群体的抗性水平。目前，已经报道的Y136F和K147Q是最常见的抗药突变基因型，前者主要与葡萄白粉菌和禾谷类白粉菌的抗药性有关，后者主要与大麦白粉菌的高抗性菌株有关；其中Y136F突变型是Délye等1997年首次在葡萄白粉菌中检测出来，随后在大麦和小麦白粉菌中也检出；而K147Q突变类型是Brown等于2005年在大麦白粉菌中首次检测到。
本申请人在扩增小麦白粉菌cyp51基因全长检测第136位氨基酸（核苷酸458）变异位点时，发现存在三种变异：野生型（A）、突变型（T），杂合型（A/T)。由于小麦白粉菌在分生孢子时期是单倍体，理论推测不可能出现杂合型变异位点，最大的可能就是菌株之间有交叉污染造成的第三种类型的变异。为明确杂合型（A/T）是否是由交叉污染造成的，我们在杂合型的菌株中挑取10个菌株（5-93，7-8，10-40，11-99，12-24，12-50，28-9，35-27，48-28，49-1）再次进行单孢子菌株的分离纯化。首先挑取单孢子堆进行扩繁，然后再挑取单孢子菌落进行扩繁，每个菌株挑取5个单孢子菌株编号，进行一代Sanger测序，结果发现10个菌株的5个纯化单孢子菌株，依然都在杂合型，说明不是污染造成的，可能是由于基因拷贝数的问题导致杂合现象存在。
无论是常规PCR，Real-time、数字PCR，还是CAPS标记不能一次性快速检测出基因cyp51第136位氨基酸上的3种变异类型，有的还需要测序（如申请CN 101397585 B），这就增加了时间和经济成本。本申请开发出KASP标记引物，一次性将cyp51基因第458核苷酸三种变异同时检测出来。通过检索，未见关于将cyp51基因第458核苷酸三种变异类型同时检测出来的报道。
发明内容
针对上述现有技术存在的不足，本发明的目的之一是提供一种小麦白粉菌抗药性高通量检测标记及其在田间抗性监测中的应用。本发明开发的KASP分子标记，适宜于高通量、高效率检测，可应用于田间抗性发生动态监测，也对正确评估田间小麦白粉菌抗性频率具有重要意义。
本申请发明人对国内采集的不同生态区100个白粉菌，随机对其中1个菌株21-2进行从头测序，剩余99个菌株重测序。利用生物信息学分析获得小麦白粉菌cyp51基因的全长，全长1696bp，两个内含子，长度分别为51bp和52bp，发现存在4个氨基酸位点的改变，对应的位点分别为S79T、F136Y、K175N、F442L。在扩增cyp51基因全长检测第136位氨基酸（核苷酸458单位）变异位点时，发现存在三种变异：野生型（A458）、突变型（T458），杂合型（A/T458)。由于小麦白粉菌在分生孢子时期是单倍体，所以不可能出现杂合型变异位点，最大的可能就是菌株之间有交叉污染造成的第三种类型的变异。为明确杂合型（A/T 458）是否是由交叉污染造成的，我们在杂合型的菌株中挑取10个菌株（5-93，7-8，，10-40，11-99，12-24，12-50，28-9，35-27，48-28，49-1）进行再次单孢子菌株的分离纯化，首先挑取单孢子堆进行扩繁，然后再挑取单孢子菌落进行扩繁，每个菌株挑取5个单孢子菌株编号，在进行一代Sanger测序，结果发现10个菌株的5个纯化单孢子菌株，依然都在杂合型，说明不是污染造成的，可能是由于基因拷贝数的问题导致杂合现象存在。
为实现针对小麦白粉菌基因cyp51第136位氨基酸处存在的三种变异的检测目的，本发明采用如下技术方案：
一种与小麦白粉菌抗药性相关的分子标记，所述分子标记位于小麦白粉菌基因cyp51，基因cyp51的核苷酸序列如SEQ ID NO:1所示，该序列长度为1696bp，该序列的第458位处存在变异，表现为野生型A、突变型T，杂合型A/T，将其标记为CYP51-458。
优选地，一种如上所述的与小麦白粉菌抗药性相关的分子标记方法，所述CYP51-458使用KASP检测，引物序列为：
Primer_AlleleFAM：GTCTTCGGGACTGATGTAGTGTA，如序列表SEQ ID NO:2所示;
Primer_AlleleHEX：GTCTTCGGGACTGATGTAGTGTT,如序列表SEQ ID NO:3所示;
Primer_Common：TTGTTCCATAATTTTGAATTAGGACAGTCA,如序列表SEQ ID NO:4所示。
优选地，如上所述的一种与小麦白粉菌抗药性相关的分子标记方法，所述KASP检测包括如下步骤：
(1)提取待测小麦白粉菌样品的DNA；
(2)取浓度为20ng/μL的模版DNA3ul,SEQ ID NO.2-4所示混合引物0.0825μL,2XMaster Mix 3μL，进行PCR扩增；
(3)采用荧光检测仪器检测PCR扩增产物的荧光信号，进行基因分型。
优选地，如上所述的一种与小麦白粉菌抗药性相关的分子标记方法，上述步骤(2)中PCR扩增的条件为：
1)94℃预变性5min；
2)94℃变性20s；
3)65℃退火30s,步骤2)-3)循环10次，每个循环退火温度降低0.8℃；
4)94℃变性20s；
5)57℃退火30s,步骤4)-5)循环38次；
6)4℃保存。
本申请的第二个目的是与小麦白粉菌抗药性相关的分子标记的应用，本申请的标记CYP51-458可用于小麦白粉菌对三唑酮抗药性基因的多态性检测。
本发明与现有技术相比有以下优点：
1）本发明获得CYP51-458位点的SNP标记，可以准确反应田间实际抗性频率，较之前报道的Y136F突变类型更为准确和客观。
2）本发明的KASP标记，具有高通量、操作简便等特点，可用于大规模田间菌株材料的高通量筛选，提高抗性监测的效率。
附图说明
图1：CYP51-458标记的KASP分型结果,靠近X轴圆点代表C(敏感菌株）基
因型，靠近Y轴圆点代表T（抗性菌株），对角线附近的圆点代表杂合C/T(抗/感菌株）基因型。
.具体实施方式：
以下是本发明的具体实施例，对本发明的技术方案做进一步描述，但本发明的内容不仅仅局限于实施例所述的范围，凡是不背离本发明构思的改变或等同替代均包括在本发明的保护范围之内。
实施例子1：小麦白粉菌的提取
1）小麦白粉菌孢子的收集温室隔离培养高感白粉病小麦苗至第一叶完全展开，剪取叶片中段，叶片正面朝上放置在琼脂保鲜培养基上，在接种装置内对上述离体叶段接种预先繁殖好的新鲜小麦白粉菌的分生孢子，接种后置于17±l℃，18 h光照条件下培养，待大量产生孢子，置于超净工作台上，将培养皿倒扣在硫酸纸上，轻轻用镊子敲打2-3次，将分生孢子收集到无菌离心管中，分生孢子生物量在100-200mg之间为最佳。
琼脂保鲜培养基配方为：水琼脂粉4克，苯并咪唑50mg,去离子水1L。
2）小麦白粉菌的DNA提取：具体提取方法如下：
①将收集的100-200mg小麦白粉菌孢子粉放入液氮预冷处理的2ml离心管中（离心管中含有3颗直径3mm不锈钢钢珠），然后在研磨器中研磨3次（具体参数：频次30下/秒，时间间隔30秒）；注意：每次研磨之前离心管都要处于液氮保护中。
②在步骤①的离心管中加入Sarcosyl(5%月桂酰肌氨酸钠溶液） 300 μL剧烈震荡30秒；加入SolutionB 700 μL继续剧烈震荡，最后置于65℃的水浴锅中15-30min；注意：可以在此步骤中取出钢珠，以免被后续的三氯甲烷污染。
SolutionB具体成分（0.2M Tris pH= 7.5，50mM EDTA，2M NaCl，2% CTAB，0.25MNa2S2O3）
③在步骤②的离心管加入氯仿600μL，剧烈震荡，14000转离心10 min（4℃），收集上清；注意：带上防护手套氯仿操作的过程中。
④在步骤③收集的上清液中加入1倍体积的预冷异丙醇，大约700μL；颠倒离心管6-8次后，14000转离心10min(4℃)，倒掉上清保留沉淀，放置冰上15min，用450μL TE（Tris-EDTA 缓冲液，10mM Tris，1mM EDTA，pH8.0）溶解沉淀。
注意：先将异丙醇加入到各管中，全部加完后再依次上下颠倒各离心管6-8次，在30℃孵育1h或4℃过夜。
⑤将步骤④溶解的溶液吸入到Amicon Ultra -0.5ml（Millipore，Ultracel型号UFC5030BK,LOTNo:R8JA97944），14000转离心30min（4℃），弃掉滤液；再加入300μL TE冲洗，直接轻微加到Amicon Ultra -0.5ml,14000 rcf 离心（4℃），将滤器倒置放到新的离心管中，1000rcf 离心1min，收集滤液。
⑥向步骤⑤的滤液中加入300μL TE和4μL RNAse到富集的DNA管中，37℃孵育1h，然后加入300μL酚-氯仿（体积比1:1）混匀6-8次，14000rcf离心10min(4℃)，转移上清到新离心管中，加入上清液2.5倍体积的无水乙醇和0.01倍体积3M醋酸钠，-20℃过夜。
⑦将步骤⑥已过夜的离心管15000rcf离心30min(4℃)，弃滤液，向离心管中加入450μL 70%乙醇，13000rcf 再次离心10min(18℃)，倒掉乙醇，在冰上干燥沉淀15min(最长不超过30min)，最后加入30-50μL TE或去离子水重悬沉淀。
质量的检测：采用Nanodrop one检测核酸质量，经过检测样品浓度≥66ng/μL（表1）
表1 60个待测样品DNA浓度检测结果
实施例子2：cyp51基因458位点
根据发明内容中找到的cyp51基因SNP位点第458位，利用Primer 5.0,将这些SNP位点转化成KASP检测标记，设计时剔除有发夹结构、引物间能形成引物二聚体、上下游引物之间Tm值超过3℃的标记，共设计16个，通过在随机检测样本中进行基因分型， 有1个KASP检测标记能够很好的区分各个体的基因型。
实施例子3：引物稀释与KASP检测引物混合
将三条引物用Tris HCL稀释到100μM后，按照体积比AlleleFAM：AlleleHEX：Common：Tris HCL=6:6:15:23的比例混合，分装后于-20℃保存，作为KASP检测引物。
实施例子4：PCR扩增体系和程序
PCR反应体系：依下表在冰上配制体系，KASP引物由（Laboratory of theGovernment Chemist 有限公司合成）
PCR反应条件:
1）94℃预变性5min；
2）94℃变性20s;
3）65℃退火30s，步骤2）-3）循环10次，每个循环退火温度降低0.8℃；
4）94℃变性20s;
5）57℃退火30s，步骤4）-5）循环38次；
6）4℃保存。
实施例子5：结果分析
利用ABI Quant Studion TM 12K Flex实时荧光定量PCR系统对扩增好的PCR体系进行SNP分布结果的收集，如图1所示，其中Y轴为抗药性基因型，X轴为敏感性基因型。对自然群体40个菌株进行检测，CYP51-458标记的KASP分型结果，靠近Y轴圆点代表C(抗性菌株）基因型4个，靠近X轴圆点代表T（敏感菌株）18个，对角线附近的圆点代表C/T(抗性/敏感菌株）基因型32个，还有6个可能是由于模版原因造成。
以上所述仅为本申请的优选实施例而已，并不用于限制本申请，对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本清的保护范围之内。
Claims (5)
1.一种检测小麦白粉菌抗三唑类杀菌剂的分子标记，其特征在于：所述分子标记位于小麦白粉菌基因cyp51，基因cyp51核苷酸序列如SEQ ID NO:1所示，该序列的第458核苷酸处存在碱基变异，表现为野生型A、突变型T，杂合型A/T，标记为CYP51-458。
2.一种如权利要求1所述的检测小麦白粉菌抗三唑类杀菌剂的分子标记方法，其特征在于：所述CYP51-458使用KASP检测，引物序列为：
Primer_AlleleFAM：GTCTTCGGGACTGATGTAGTGTA，如序列表SEQ ID NO:2所示;
Primer_AlleleHEX：GTCTTCGGGACTGATGTAGTGTT,如序列表SEQ ID NO:3所示;
Primer_Common：TTGTTCCATAATTTTGAATTAGGACAGTCA,如序列表SEQ ID NO:4所示。
3.如权利要求2所述的一种检测小麦白粉菌抗三唑类杀菌剂的分子标记方法，其特征在于：所述KASP检测包括如下步骤：
(1)提取待测小麦白粉菌样品的DNA；
(2)取浓度为20ng/μL的模版DNA3ul,SEQ ID NO.2-4所示混合引物0.0825μL,2XMasterMix 3μL，进行PCR扩增；
(3)采用荧光检测仪器检测PCR扩增产物的荧光信号，进行基因分型。
4.如权利要求3所述的一种检测小麦白粉菌抗三唑类杀菌剂的分子标记方法，其特征在于：所述步骤(2)中PCR扩增的条件为：
1)94℃预变性5min；
2)94℃变性20s；
3)65℃退火30s,步骤2)-3)循环10次，每个循环退火温度降低0.8℃；
4)94℃变性20s；
5)57℃退火30s,步骤4)-5)循环38次；
6)4℃保存。
5.一种如权利要求1所述的分子标记的应用，其特征在于：所述标记CYP51-458可用于小麦白粉菌对三唑酮抗药性基因的多态性检测。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211404362.8A CN116411112A (zh) | 2022-11-10 | 2022-11-10 | 一种检测小麦白粉菌抗三唑类杀菌剂的分子标记方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211404362.8A CN116411112A (zh) | 2022-11-10 | 2022-11-10 | 一种检测小麦白粉菌抗三唑类杀菌剂的分子标记方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116411112A true CN116411112A (zh) | 2023-07-11 |
Family
ID=87055376
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211404362.8A Pending CN116411112A (zh) | 2022-11-10 | 2022-11-10 | 一种检测小麦白粉菌抗三唑类杀菌剂的分子标记方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116411112A (zh) |
-
2022
- 2022-11-10 CN CN202211404362.8A patent/CN116411112A/zh active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3258658B2 (ja) | ユニバーサル ユーバクテリア核酸プローブ及び方法 | |
| Weising et al. | DNA fingerprinting in plants and fungi | |
| US6733965B2 (en) | Microsatellite DNA markers and uses thereof | |
| EP1766083B1 (en) | Method for the detection of fusarium graminearum | |
| CN101492743A (zh) | 一种病原性真菌检测基因芯片 | |
| WO1993004202A1 (en) | Polynucleotide probes for salmonella | |
| Chtourou-Ghorbel et al. | Comparative genetic diversity studies in the genus Lathyrus using RFLP and RAPD markers | |
| US8377656B2 (en) | Compositions and methods for detecting Cryptococcus neoformans | |
| US20110287965A1 (en) | Methods and compositions to detect clostridium difficile | |
| CN110846433B (zh) | 一种与小麦白粉菌抗药性相关的kasp标记及其应用 | |
| US20030113722A1 (en) | Detection of fusarium species infecting corn using the polymerase chain reaction | |
| US9458514B2 (en) | Nucleic acids probes for detection of yeast and fungal | |
| Miyazaki et al. | Specific PCR assays for the detection of Trichoderma harzianum causing green mold disease during mushroom cultivation | |
| KR20120045917A (ko) | 벼키다리병균(Ｆｕｓａｒｉｕｍ ｆｕｊｉｋｕｒｏｉ) 신속 검출용 특이 프라이머 및 이를 이용한 균 검출방법 | |
| CN116411112A (zh) | 一种检测小麦白粉菌抗三唑类杀菌剂的分子标记方法 | |
| US7794982B2 (en) | Method for identifying gene with varying expression levels | |
| JP2014003946A (ja) | サーモアスカス属真菌の検出方法 | |
| CN113046466B (zh) | 一组与小麦白粉病抗性显著关联的snp位点及其在遗传育种中的应用 | |
| Sagar et al. | Determination of genetic variations among different Trichoderma isolates using RAPD marker in Bangladesh | |
| CN112662804B (zh) | 一种检测稻瘟病菌无毒基因AvrPi9致病性变异的引物组、试剂盒及方法 | |
| JP5548385B2 (ja) | 耐熱性菌類の検出方法 | |
| WO2001023616A2 (en) | Method of identifying pathogenic cryptococci | |
| CN113604602B (zh) | 捕食线虫真菌寡孢节丛孢的str分子标记 | |
| CN117757983B (zh) | 与硬粒小麦抗白粉病基因PmYD001共分离的分子标记的引物及其应用 | |
| CA2381204A1 (en) | Pcr-based detection and quantification of tapesia yallundae and tapesia acuformis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |