CN113789404A - Functional specific molecular marker of rice blast resistance gene Pik-zh and application - Google Patents

Functional specific molecular marker of rice blast resistance gene Pik-zh and application Download PDF

Info

Publication number
CN113789404A
CN113789404A CN202111045221.7A CN202111045221A CN113789404A CN 113789404 A CN113789404 A CN 113789404A CN 202111045221 A CN202111045221 A CN 202111045221A CN 113789404 A CN113789404 A CN 113789404A
Authority
CN
China
Prior art keywords
pik
rice blast
rice
resistance gene
seq
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.)
Granted
Application number
CN202111045221.7A
Other languages
Chinese (zh)
Other versions
CN113789404B (en
Inventor
杨德卫
何旎清
郑向华
黄凤凰
程朝平
叶宁
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rice Research Institute Fujian Academy Of Agricultural Sciences
Original Assignee
Rice Research Institute Fujian Academy Of Agricultural Sciences
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Rice Research Institute Fujian Academy Of Agricultural Sciences filed Critical Rice Research Institute Fujian Academy Of Agricultural Sciences
Priority to CN202111045221.7A priority Critical patent/CN113789404B/en
Publication of CN113789404A publication Critical patent/CN113789404A/en
Application granted granted Critical
Publication of CN113789404B publication Critical patent/CN113789404B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • C12Q2600/13—Plant traits
    • 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

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Mycology (AREA)
  • Botany (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a functional specificity molecular marker of a rice blast resistance gene Pik-zh and application thereof, wherein the functional specificity molecular marker of the rice blast resistance gene Pik-zh is Pik-zh-InDel, and a molecular marker which is in a specific banding pattern with the rice blast resistance gene Pik-zh is amplified from a genome DNA of a rice variety carrying the rice blast resistance gene Pik-zh through a primer pair SEQ ID NO.1 and SEQ ID NO. 2; wherein SEQ ID NO.1 is 5'-AGCAGTGGGACGAGTGGGA-3'; 5'-GGTAGGACACCTGCCATCA-3' for SEQ ID NO. 2; the functional specificity molecule of the rice blast disease-resistant gene Pik-zh obtained by the invention can be applied to identifying the rice blast disease-resistant gene in rice varieties.

Description

Functional specific molecular marker of rice blast resistance gene Pik-zh and application
Technical Field
The invention belongs to the technical field of biomolecular markers, and particularly relates to a functional specificity molecule of a rice blast resistance gene Pik-zh and application thereof.
Background
Rice is one of the major food crops in the world, and rice blast caused by rice blast germs is a worldwide disease and has a tendency to increase in recent years. The rice blast not only causes great yield reduction (10% -35%) of rice, but also influences the quality of rice when the rice is not harvested even if the rice is severe. From the viewpoint of grain safety and rice disease-resistant breeding, breeding of disease-resistant varieties is one of the most economical and effective methods for preventing and treating rice blast. But the traditional disease-resistant breeding strategy has the characteristics of large workload, long period and the like. Therefore, there is an urgent need to change the traditional strategy of breeding for disease resistance.
The DNA molecular marker is a genetic marker developed on the basis of polymorphism of biomacromolecules, and is currently applied to aspects such as genome mapping, gene localization, genetic diversity evaluation, functional genomics research, transgenic plant identification, molecular marker-assisted selective breeding and the like. The gene function specific molecular marker is a molecular marker developed by utilizing the specificity and the difference of DNA sequences in a target gene. The gene characteristic molecular marker is a functional molecular marker developed from functional SNP loci or InDels in a target functional gene sequence, can accurately track and locate functional alleles, has higher accuracy in the identification of crop phenotypic traits, and can efficiently screen beneficial genes required by breeding.
In the 60 s of the 20 th century, the research work of rice blast resistance gene analysis of rice varieties is first developed in Japan, 14 genes on the first 8 resistance sites are identified, and a set of identification system for rice blast resistance gene analysis is established. However, some disease-resistant genes are located at the same site, and the multiple genes and the functional sequence and the non-functional sequence are highly homologous, so that the traditional method is difficult to distinguish. Therefore, by analyzing the specific sequence difference between the target gene and the allele and developing the function specific molecular marker to select the target gene, the selection reliability is high, and the breeding pace is greatly accelerated.
As the Pik locus located at the long arm end of the 11 th chromosome contains more than 5 alleles Pik-m, Pik-p, Pik-h, Pik-s and the like with different resistance profiles and race specificities, the locus has been the hot door for the research on rice blast resistance, wherein Pik-m, Pik-p, Pik-h and Pik-s have been successfully identified and cloned, and related function specific molecular markers of the alleles have been gradually developed and applied.
The rice blast resistance gene Pik-ZH is a new main disease-resistant gene which is identified from a rice variety ZH11 (Zhonghua 11) by the applicant and has the same allele with Pik, the total length of CDs is 3438bp, the detailed nucleic acid sequence is shown in SEQ ID NO.4 by using a map-based cloning method, and further analysis on the rice blast resistance gene Pik-ZH discovers that the Pik-ZH sequence has specific difference with partial sequences of Pik, Pik-s, Pik-h and Pik-p which are cloned at the site at present, and the Pik-ZH gene can show stronger disease-resistant reaction on strains D527 and KJ201 in China. Therefore, in order to more accurately and effectively apply the broad-spectrum resistance gene Pik-zh of rice blast to rice resistance breeding work, the development of a Pik-zh specific molecular marker which truly reflects a target gene and is convenient and easy to use is urgently needed.
Disclosure of Invention
The invention provides a functional specific molecular marker and application of a rice blast resistance gene Pik-zh, creatively discovers the Pik-zh specific molecular marker which can truly reflect the broad-spectrum resistance gene Pik-zh of rice blast, and simultaneously, the Pik-zh specific molecular marker which is convenient and easy to use and has higher accuracy and stability is prepared, so that the broad-spectrum resistance gene Pik-zh of rice blast is better applied to rice resistance breeding.
The purpose of the invention is realized by the following technical scheme:
one of the purposes of the invention is to provide a functional specific molecular marker of a rice blast resistance gene Pik-zh, the functional specific molecular marker of the rice blast resistance gene Pik-zh is Pik-zh-InDel, and a molecular marker which is in a specific banding pattern with the rice blast resistance gene Pik-zh is amplified from the genome DNA of a rice variety carrying the rice blast resistance gene Pik-zh through a primer pair SEQ ID NO.1 and SEQ ID NO. 2; wherein, the nucleotide sequences of the primer pair SEQ ID NO.1 and SEQ ID NO.2 are as follows:
SEQ ID NO.1：5’-ACGAGTGGGATCGATACTG-3’；
SEQ ID NO.2：5’-GGTAGGACACCTGCCATCA-3’；
the rice variety carrying the rice blast resistance gene Pik-zh is a rice disease-resistant variety Zhonghua 11.
Further, the rice blast resistance gene Pik-zh comprises a gene segment SEQ ID NO.3 partially coding NBS-LRR type protein.
One of the purposes of the invention is also to provide a preparation method of the functional specific molecular marker of the rice blast resistance gene Pik-zh, which comprises the following steps:
(1) sequence comparison is carried out aiming at the Pik-zh locus of the rice blast resistance gene, and the specific insertion/deletion InDel locus of the Pik-zh and other rice blast allelic resistance genes which can be distinguished from the locus is screened;
(2) designing a gene specific primer at the InDel site by utilizing the InDel site information obtained in the step (1);
(3) carrying out PCR amplification by taking the total DNA of a rice blast resistant variety carrying the rice blast resistant gene Pik-zh as a template, wherein the obtained PCR product is the specific molecular marker Pik-zh-InDel of the rice blast resistant gene Pik-zh.
One of the purposes of the invention is to apply the functional specific molecular marker of the rice blast resistance gene Pik-zh to the identification of rice blast resistance genes of rice varieties.
The invention also provides a kit for identifying rice blast resistance of rice varieties, which comprises a primer pair with nucleic acid sequences shown as SEQ ID NO.1 and SEQ ID NO. 2.
The invention has the beneficial effects that:
1. the invention is a functional specific molecular marker developed aiming at a new major disease-resistant gene Pik-zh which is allelic with Pik, and the functional specific molecular marker of Pik-zh has the advantages of low cost, high efficiency, high accuracy and the like in practical application; the Pik-zh functional specific molecular marker is Pik-zh-InDel, and Pik-zh can be successfully distinguished from other non-Pik-zh genes on the site by using an electrophoresis detection method, so that the detection efficiency is high; meanwhile, the Pik-zh specific molecular marker Pik-zh-InDel provided by the invention is a dominant marker, exists in the Pik-zh gene, and has a theoretical value of 100 percent of screening capacity on Pik-zh
2. The Pik-zh functional specificity molecular marker provided by the invention is Pik-zh-InDel, only PCR is combined with agarose gel electrophoresis or non-denatured polyacrylamide gel electrophoresis, the cost is low, the accuracy rate is high, the flux is high, in addition, the specificity is high, and the Pik-zh functional specificity molecular marker is particularly suitable for agricultural production practice and germplasm resource screening, transgenic breeding, gene polymerization and MAS technology-based resistance breeding under any genetic background, and greatly improves the breeding efficiency and the accuracy.
Drawings
FIG. 1 shows the amplification of 6 selected representative varieties of total DNA as templates using primers F1 and R1 shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments and the accompanying drawings, wherein the embodiments are only for explaining the present invention and do not limit the present invention in any way.
Example 1
The preparation method of the functional specific molecular marker of the rice blast resistance gene Pik-zh specifically comprises the following steps:
(1) analysis of Pik-zh insertion/deletion (InDel) sites:
downloading genome sequences of different alleles of Pik loci and corresponding loci of different rice varieties from a public database: pik-m (GenBank: GU811860.1), Pi1 (GenBank: HQ606329.1), Pik (GenBank: AB616658.1), Pik-h (GenBank: HQ662330.1), Pik-s (GenBank: HQ662329.1), Pik-p (GenBank: HM035360.1), Shuhui 498 (GenBank: CP018167.1), Nippon (Locus: LOC _ Os11g42010), then carrying out sequence alignment on the Pik-zh Locus of the rice blast resistance gene in a rice database, carrying out design of specific markers according to the functional region difference Locus, sequencing the genome sequence of the rice donor varieties Nippon, Shuhui 498 and Pik allele which have no complete corresponding region, wherein the missing and different base parts in the alignment result are identified InDel loci, and the alignment result is as follows: pik-zh:
Figure BDA0003250960000000031
Shuhui498：
Figure BDA0003250960000000032
Figure BDA0003250960000000041
Pik-m：
Figure BDA0003250960000000042
Pi1：
Figure BDA0003250960000000043
Pik：
Figure BDA0003250960000000044
Pik-h：
Figure BDA0003250960000000045
Pik-s：
Figure BDA0003250960000000046
Pik-p：
Figure BDA0003250960000000047
deletion of the corresponding sequence in Nipponbare
Figure BDA0003250960000000048
Wherein, the boxed part is the single base part of the Pik-zh gene with difference detected after the comparison, and the blank is the single base part of the missing Pik-zh gene detected after the comparison, further analysis shows that a section of sequence in the Pik-zh gene is extremely specific, and has 9bp difference compared with the corresponding sequences of Pik-m, Pi1, Pik-h, Pik-s, Pik-p and Shuhui 498, while compared with the corresponding sequence of Nipponbare, the partial sequence of Nipponbare is completely missing.
(2) Designing a gene specific primer at the InDel site by using the InDel site information obtained in the step (1):
according to the design principle of InDel markers, primer design is carried out at the InDel differential site, and the base sequences of primer pairs are shown as follows: SEQ ID NO.1 (F1): 5'-ACGAGTGGGATCGATACTG-3', respectively;
SEQ ID NO.2(R1)：5’-GGTAGGACACCTGCCATCA-3’；
(3) carrying out PCR amplification by taking the total DNA of a rice blast resistant variety carrying a rice blast resistant gene Pik-zh as a template:
in this example, 6 representative rice varieties were selected, in order: 11 of Zhonghua, 498 of Shuhui, CO39, Nipponbare, Lijiang Xinjiang black valley and Tetepu;
performing conventional PCR amplification by using the primers SEQ ID NO.1 and SEQ ID NO.2 and the total DNA of the 6 rice varieties as templates to obtain a PCR product, namely a rice blast resistance gene Pik-zh gene specific molecular marker Pik-zh-InDel;
wherein, the PCR amplification reaction system comprises the following steps:
2x Reaction Mix:16μL；
primer SEQ ID NO.1(F1) (10 μm): 1 mu L of the solution;
primer SEQ ID NO.1(F1) (10 μm): 1 mu L of the solution;
2 μ L of DNA template (20-50 ng/. mu.L);
ddH2o: make up to 25 μ L;
the PCR temperature cycling conditions were as follows: 5 minutes at 94 ℃; 30 seconds at 94 ℃; 30 seconds at 55 ℃; 30 seconds at 72 ℃; 35 cycles; 7 minutes at 72 ℃; storing at 10 deg.C;
after the PCR reaction is finished, 10-20ul of PCR sample products are subjected to electrophoresis detection on 3% agarose for 40 minutes under the electrophoresis condition of 110V, and after the electrophoresis is finished, a gel imaging system is used for photographing to observe the electrophoresis result, wherein fragments containing the expected target size are positive, and fragments containing or not containing the target size are negative.
Example 2
The application of the functional specific molecular marker of the rice blast disease-resistant gene Pik-zh in identifying the rice blast resistant genes of rice varieties is that firstly, a primer F1 shown in SEQ ID NO.1 and a primer R1 shown in SEQ ID NO.2 are utilized to perform PCR amplification on genomes of 6 rice varieties to be detected; secondly, detecting by using agarose electrophoresis, wherein if a nucleotide fragment with the molecular weight of 108bp in a PCR product is detected, the rice variety to be detected carries the rice blast resistance gene Pik-zh; if the nucleotide fragment with the molecular weight of 108bp is not detected, the rice variety to be detected does not carry the rice blast resistance gene Pik-zh.
Referring to FIG. 1, the amplification of the total DNA of 6 selected representative varieties using the primer F1 shown in SEQ ID NO.1 and the primer R1 shown in SEQ ID NO.2 provided in example 1 as templates; referring to FIG. 1, lane 1 is DNA ladder, lane 2 is the fragment obtained by PCR using the genome of flower 11 in the rice variety of Pik-zh donor variety as a template, and is the specific banding pattern of the rice blast resistance gene Pik-zh, i.e., lane 2 is the specific molecular marker Pik-zh-InDel of Pik-zh gene, and the fragment size is 108 bp; the DNA templates in lanes 3-7 are Shuhui 498, CO39, Nipponbare, Lijiang New Yougu, and Tetepl in that order; as shown in attached figure 1, in the rice variety carrying the Pik-zh gene, an electrophoresis detection strip of a PCR amplification product is shown by 108bp, the Zhonghua 11 and the Tepu both contain the Pik-zh gene but do not contain a functional gene of the site, the 108bp strip cannot be detected by electrophoresis, and the Shuhui 498, the CO39, the Nipponbare and the Lijiang Xinjiang black valley do not contain the Pik-zh gene.
As shown in the attached figure 1, the test result is identical with the design analysis, and the functional specificity molecular marker of the resistance gene Pik-zh can identify the rice blast resistance gene Pik-zh, so that the method is suitable for agricultural production practice and germplasm resource screening, transgenic breeding, gene polymerization and MAS technology-based resistance breeding of Pik-zh under any genetic background, and greatly improves the breeding efficiency and accuracy.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Sequence listing
<110> Rice research institute of agricultural science institute of Fujian province
<120> functional specific molecular marker of rice blast resistance gene Pik-zh and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
agtgggacga gtgggatcg 19
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggtaggacac ctgccatca 19
<210> 3
<211> 173
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gggagcagtg aaaacattgc gggcagcagt gggacgagtg ggatcgatac tgagcctgca 60
caggcacagc atgataatct ccctgctgtt cgagatgact acaagggaaa agggattctt 120
cttgatggca ggtgtcctac ctgcggccga gcgactaaaa ttgaagagga aac 173
<210> 4
<211> 6359
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atggaggcgg ctgccatggc cgtaaccgca gccacggggg ccttggcgcc cgtgctagtg 60
aagctggccg ctttgctgga cgacggggag tgcaatcttc tggaggggag ccggagcgac 120
gcagagttca tcagatccga gctggaggcc gttcattctc tcctcacccc aaatatcttg 180
gggaggatgg gggatgacga tgcggcgtgc aaggatggct tgattgcgga ggtccgggag 240
ctgtcctacg acctggatga tgccgtcgac gacttcttgg agctcaattt cgagcagcga 300
agaagcgcaa gccctttcgg tgagctcaag gcaagagttg aggagcatgt ctccaatcgc 360
ttctctgact ggaagctacc ggcggcgagc cttccgccgt cgtcggtaca ccgccgagct 420
ggcttgccgc caccagatgc agagctggtg gggatggaca aacgtatgga agagctcacc 480
aaattgctgg aacaagggag caatgatgct tcacgatggc gcaagcgaaa accgcatttc 540
ccgctcagaa aaacagggct aaaggtacgg ttggatctcg attcttcaca aatatagctt 600
cctccgatag cagtgccaac gaatttgttt acttctctct tgattcttta atttggaagt 660
actgtataac aaacatggag ggatcgtcat ttaacttaat tttttttgtt gcagcaaaaa 720
atcgtgatca aggttgccat ggagggcaac aattgccgtt caaaagcaat ggctttagtt 780
gcgagcactg gaggagtgga ctcggttgcg ctcgcaggtg atctaagaga caagatagag 840
gtggtcggtt atggcattga ccccatcaag ctgatctccg cgctccggaa gaaggtgggc 900
catgcggagt tgctgcaggt cagccaagca aaaaaagatg tgaaggagac gacgccgatg 960
cttgcgccgg tgaaatccat atgtgaattt cacaaggtca aaacagtttg catccttgga 1020
ttgccaggtg gaggcaaaac aacggttgcc agagaattat atgacgcctt gggaacgcac 1080
ttcccatgcc gggttttcgt gtcagtctct ccaagttcta gtcccagtcc caatctcaca 1140
aagactcttg cagacatttt cgctcaagca caactaggag taactgatac acttagcaca 1200
ccatatggtg ggagtgggac cgggagagct cttcaacaac atctcatcga caacatatca 1260
gctttcctcc tcaacaaaaa gtaagcaata acttatatct cttatctagc ttgtccttaa 1320
tccggcttcc caaattaaag tgaacaggct atatgtccat ataatgttgg gttttttttt 1380
tctgtcctcg atgaggtata tatcaagata tgccaaactt tttttaatta gcgttacttt 1440
attctaccta gtaaatagat acataaatgg ggcaggcccc tcctttgtga caggccaatg 1500
gttttatgat gtggaaacaa ctcccttatc actgaagctc ttgctattac tcgtgatggt 1560
actgatcttg caaaggcttg cagtatttgt aaatccacag ggagtggtca tctttttttt 1620
ttaagataat gaatagaaat ctggcctcta tatagaaagc caaaggttac acaagtgcat 1680
acactccaaa tctcaaagct gagaaacaca aaaaacaaac gaccaaaaag aaaagacgat 1740
aagattaagt ttcaagcttt tcctccttgc ttcaaaaaac agggaaatgc agagctagcc 1800
cgctcttccc ttgccatgcc ctctcactgc cattgcaact accacaaaag ctcccctgtt 1860
tcgccgtgag cgaagggagg ggatggaact agtctgccgc tttgttgtcg ccaagcactg 1920
gtttcatgct gcccaaacaa cctatgcccc gctagcgata acgagtctgt tgccgccatg 1980
ctgttgtcgc tggagagcac cacttgtcgc tgctcagcta tgtgttaagt aagctactaa 2040
caatctctcc attctcttgt agactgctta taaaagattg tacttcaact tttgattaat 2100
gaaatagagt tcctgcctta caacaacaag aacgagagag agagagagag ccaatatcac 2160
gagttagcac tgtgaagact tcctgactgt tatgtcttca agaacagtat aaaagcattt 2220
gccactataa aaatatgata gccctcaaga tcattaaaaa tatgtcatgt tcttttacct 2280
tcataacaac cagtatcaca aatatgaaaa catttaaagg aaaatcagta tttgccattt 2340
atgtaaagaa aaactagact agaactctgg gcttttattt tagtgcacac tgtatacatg 2400
tgtgttcttt gacgtatata cttgatatgt tttcaaaaac ttttgggggc atatctgtaa 2460
cccatgcccc atgcatccaa agtagtagtg agacaacttt cgtatcaatg gacatgtggc 2520
attaaattta atcccaatat ttgttctctc gaagtcctga acatataatt ggacaaaatt 2580
atataattta attaggccaa ttgagccata tatacacaca ctataagaaa cttctacaaa 2640
acaaacattc gctcctttta ggttttgacc agtctataca tgaaccagcc aacggtgtgc 2700
tggtctgact ataaatgttt ttttagagag taaaggcacc ttcaagtacc tctctcgagg 2760
acagtaaaaa cacccatgct ttaaggttat gaacaacaaa gcatagaaca accacaagaa 2820
aaaaaaacaa ctagaacaag ttctggaact agttagggat tcgggaagcc attgcttgct 2880
ggcttgggac aagctatgcc tgccattgga ctatggtgga gactgggcat aaaggatctc 2940
aatctcttgg agattggcct atgtcctagg tggccatggc tgactgcaag gagatatatt 3000
tcttgcaacg gaaattttca ttgcaaaagc tgaaaatcgt tgcgatatgt acctattgtg 3060
acataattga atccattggg agccgtagca ataaatcccg tggtaatagg ctattgcaat 3120
gattttccga tggttgcata aaaaggtgct attacaatgg ttttgcattg ctattgcaac 3180
aacttgatat gtcataggta tttattgcaa cagtttgtac catgctgcaa taagatttcg 3240
caacacagac ctattcatta aatagcctat ttccacacat tcatttttgt ggcaataatt 3300
cagtgcgaca agcatgttct atagctataa cctgatgcaa cgggttagac ttgttgcagt 3360
aacttattag cacatttaat tttttactaa atttatattg attcaactat ttcaacatca 3420
ttcaatacag taaatactgg aaaatgcaag caatctccaa aaattcacca ataagagaaa 3480
cgaattaaat catatacaca attatttcac accgtacagt gcacttaagt gtaggaaata 3540
taatgctata tacatttcca atcctcaata caggttctgt aaataatgaa aagaaacgtc 3600
aatttgattt cttgaccggt ccctctgaga cacacagtgc tttcctcagc aacatcgttg 3660
ccgatctatg taacttgtaa actccttgtg cttaataaat tctggccagg gttacttcag 3720
cctgcatggc gaaaaaaaag aacttgttag ggagatcgca aacaaatgtt agctgcccca 3780
tttcttctct actgaccata aaagctctag gatcaggtca tgaaaacaat gccgtcagac 3840
cggaaatact caagattcag aacacgactc ctaggaactg caagtttaaa tttcaacaaa 3900
agcaaaatac aaccagatga caaactaata tatatcagct gactgttcat ctcaagtgga 3960
ctcacatctg tgaaattgtt agttctatgc atatatgcta atgtaatact aatatgctat 4020
tgaggcttat tactcttctc gacttttata ggtatctcat tgtaatcgat gacatttggc 4080
attgggaaga atgggaagtc atcagaaagt ccattcccaa gaatgatctg ggtggtagaa 4140
taatcatgac tactcgtctt aattcaatag ctgagaagtg ccacactgat gacaatgatg 4200
tttttgtcta cgaagttggg gatctagata ataatgatgc tttgtcgttg tcttggggga 4260
tagcaacaaa gtctggggca ggcaacagga tcggaactgg agaggataat ccatgctatg 4320
atattgtgaa catgtgttat ggtatgcctt tagcacttat ttggctgtcg tcagcattgg 4380
ttggagagat agaagaatta ggtggtgctg aagtgaaaaa atgtagggat ttgagacaca 4440
tagaggatgg tattttggac atcccatcct tacaaccatt ggcggagagt ttatgccttg 4500
gttataacca tcttcctctt tatctgagga ctttgttgtt gtactgtagt gcataccatt 4560
ggtctaacag aatcgaaagg ggtcgtctgg tcaggaggtg gattgcggaa ggatttgtgt 4620
cggaagagaa agaagcagaa ggttactttg gcgagcttat tgacagagga tggattacgc 4680
agcacggaga caacaacagt tataattact atgagatcca ccccgtgatg ctggccttcc 4740
tgagatgcaa gtccaaggag tacaattttt taacatgctt gggtctggga tctgatacta 4800
gtactagtgc atcctcccca aggttgattc gccggctgtc tcttcagggg gggtatccag 4860
tggactgctt gtcaagcatg agtatggatg tgtcacacac ttgcagcctt gtcgtccttg 4920
gtgacgtggc gcgatccaag ggaatcccct tctatatgtt taagcgcttg cgagtgttgg 4980
accttgaaga taataaggat atacaggatt ctcatctgca gggcatatgt gaacagttaa 5040
gcctcagagt gaggtacctt ggtctcaagg gaacgcggat ccgaaagctc cctcaggaga 5100
tgaggaagct gaagcatttg gagattttgt atgtggggag cactcggatc agtgaacttc 5160
cgcaagagat tggagagctg aagcatctgc ggattctgga cgtgagaaac acggacatca 5220
ctgagctccc actgcagata cgggagctgc agcatctgca cactctggac gtgaggaaca 5280
cgccaatcag tgagctcccg ccgcaggttg gcaagctgca gaatctcaag attatgtgcg 5340
tgaggagcac tggggttagg gagctcccaa aggagattgg ggagctgaat catctacaga 5400
ctctggacgt gagaaacacg agggtgagag agctgccatg gcaagctggc cagatctccc 5460
aatcgttgcg cgtgcttgcc ggtgacagtg gcgatggcgt gcggttgccc gaaggcgtct 5520
gcgaagctct gatcaacggt attccagggg ctacgcgtgc aaaatgcagg gaggttctgt 5580
ccatcgcgat catcgatcgt ttcggacctc cccttgttgg gatattcaaa gttcccggca 5640
gtcatatgcg tatcccgaag atgatcaaag accacttccg cgttctttct tgcctagaca 5700
tcaggctctg ccacaagctt gaggatgatg accaaaagtt cctcgccgag atgcccaacc 5760
tgcagacgct cgtgctgagg ttcgaggccc taccaagaca acccataacc atcaacggca 5820
caggcttcca gatgctggag agcttccgtg tcgacagccg ggtgccaagg atagccttcc 5880
atgaagacgc catgcccaac ctcaagcttc tcgagttcaa gttctacgcc ggcccagcaa 5940
gcaacgatcc catcggcatc accaacctga agagcctcca aaaggtggtc tttcggtgct 6000
cgccatggta caagagcgac gcccctggca tcagcgccac cattgacgtc gtgaagaaag 6060
aagccgagga gcatcccaac cggccgatca ccctcctcat caatgctggg tataaggaga 6120
tatcaattga gtcacatggg agcagtgaaa acattgcggg cagcagtggg acgagtggga 6180
tcgatactga gcctgcacag gcacagcatg ataatctccc tgctgttcga gatgactaca 6240
agggaaaagg gattcttctt gatggcaggt gtcctacctg cggccgagcg actaaaattg 6300
aagaggaaac ccaagatcga gtagcagata ttgaaattca aacagaaact actagctag 6359

Claims (5)

1. A functional specific molecular marker of a rice blast resistance gene Pik-zh is characterized in that: the rice blast resistance gene Pik-zh functional specific molecular marker is Pik-zh-InDel, and a molecular marker which is in a specific banding pattern with the rice blast resistance gene Pik-zh is amplified from the genome DNA of a rice variety carrying the rice blast resistance gene Pik-zh through a primer pair SEQ ID NO.1 and SEQ ID NO. 2; wherein, the nucleotide sequences of the primer pair SEQ ID NO.1 and SEQ ID NO.2 are as follows:
SEQ ID NO.1：5’-ACGAGTGGGATCGATACTG-3’；
SEQ ID NO.2：5’-GGTAGGACACCTGCCATCA-3’；
the rice variety carrying the rice blast resistance gene Pik-zh is a rice disease-resistant variety Zhonghua 11.
2. The functionally specific molecular marker of the rice blast resistance gene Pik-zh according to claim 1, characterized in that: the rice blast resistance gene Pik-zh comprises a gene segment SEQ ID NO.3 of a part of coded NBS-LRR type protein.
3. The method for producing a function-specific molecular marker for the rice blast resistance gene Pik-zh according to any one of claims 1 to 2, comprising the steps of:
(1) sequence comparison is carried out aiming at the Pik-zh locus of the rice blast resistance gene, and the specific insertion/deletion InDel locus of the Pik-zh and other rice blast allelic resistance genes which can be distinguished from the locus is screened;
(2) designing a gene specific primer at the InDel site by utilizing the InDel site information obtained in the step (1);
(3) carrying out PCR amplification by taking the total DNA of a rice blast resistant variety carrying the rice blast resistant gene Pik-zh as a template, wherein the obtained PCR product is the specific molecular marker Pik-zh-InDel of the rice blast resistant gene Pik-zh.
4. The use of the rice blast resistance gene Pik-zh of any one of claims 1 to 2 as a functional specific molecular marker for identifying rice blast resistance genes of rice varieties.
5. A kit for identifying rice blast resistance of a rice variety, characterized in that: comprises a primer pair with nucleic acid sequences shown as SEQ ID NO.1 and SEQ ID NO. 2.
CN202111045221.7A 2021-09-07 2021-09-07 Functional specificity molecular marker of rice blast resistance gene Pik-zh and application Active CN113789404B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111045221.7A CN113789404B (en) 2021-09-07 2021-09-07 Functional specificity molecular marker of rice blast resistance gene Pik-zh and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111045221.7A CN113789404B (en) 2021-09-07 2021-09-07 Functional specificity molecular marker of rice blast resistance gene Pik-zh and application

Publications (2)

Publication Number Publication Date
CN113789404A true CN113789404A (en) 2021-12-14
CN113789404B CN113789404B (en) 2023-11-17

Family

ID=79182914

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111045221.7A Active CN113789404B (en) 2021-09-07 2021-09-07 Functional specificity molecular marker of rice blast resistance gene Pik-zh and application

Country Status (1)

Country Link
CN (1) CN113789404B (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105950747A (en) * 2016-06-02 2016-09-21 福建省农业科学院生物技术研究所 Rice blast resistance gene Pi1 functional specificity molecular marker and application thereof
CN107937600A (en) * 2018-01-19 2018-04-20 福建省农业科学院生物技术研究所 A kind of rice blast resistance gene seat Pik functional genes molecular labeling and its application
CN111978387A (en) * 2020-08-26 2020-11-24 武汉大学 Rice blast resistance gene Pikg, encoding protein and application thereof
CN113106104A (en) * 2021-04-20 2021-07-13 福建农林大学 Rice blast resistance related gene OsNAC29 and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105950747A (en) * 2016-06-02 2016-09-21 福建省农业科学院生物技术研究所 Rice blast resistance gene Pi1 functional specificity molecular marker and application thereof
CN107937600A (en) * 2018-01-19 2018-04-20 福建省农业科学院生物技术研究所 A kind of rice blast resistance gene seat Pik functional genes molecular labeling and its application
CN111978387A (en) * 2020-08-26 2020-11-24 武汉大学 Rice blast resistance gene Pikg, encoding protein and application thereof
CN113106104A (en) * 2021-04-20 2021-07-13 福建农林大学 Rice blast resistance related gene OsNAC29 and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HAO LIU等: "NBS-LRR Protein Pik-H4 Interacts with OsBIHD1 to Balance Rice BlastResistance and Growth by Coordinating Ethylene-BrassinosteroidPathway", 《FRONT. PLANT SCI》 *
WU，J等: "GeneBank：AP018855.1", 《NIBI:GENEBANK》 *

Also Published As

Publication number Publication date
CN113789404B (en) 2023-11-17

Similar Documents

Publication Publication Date Title
Chapman et al. A genomic scan for selection reveals candidates for genes involved in the evolution of cultivated sunflower (Helianthus annuus)
Yang et al. Application of next-generation sequencing for rapid marker development in molecular plant breeding: a case study on anthracnose disease resistance in Lupinus angustifolius L.
Li et al. Comparative mapping of quantitative trait loci associated with waterlogging tolerance in barley (Hordeum vulgare L.)
CN103305510B (en) Rice blast resistance gene Pi9 gene specificity molecular marker Pi9SNP as well as preparation and application thereof
CN105385697B (en) Sesame inflorescence finite basis is because of Sidt1 and its SNP marker
KR101095220B1 (en) DNA marker associated with resistance of cabbage clubroot disease and uses thereof
CN105950747A (en) Rice blast resistance gene Pi1 functional specificity molecular marker and application thereof
CN110551844A (en) Sugarcane cultivar genome SSR molecular marker development method and application
Lee et al. Construction of a plant-transformation-competent BIBAC library and genome sequence analysis of polyploid Upland cotton (Gossypium hirsutum L.)
CN108977568B (en) Rice blast resistance gene Pik-p functional specificity molecular marker and application thereof
CN112176083B (en) Functional molecular marker of wheat powdery mildew resistance related gene Pm41 and application thereof
Lee Development and evolution of molecular markers and genetic maps in capsicum species
Singh et al. Exome sequencing and advances in crop improvement
CN104928299B (en) Leaf Spot Caused by Corynespora cassiicola on Cucumber disease-resistant gene Cca and its encoding proteins and application
CN113789404B (en) Functional specificity molecular marker of rice blast resistance gene Pik-zh and application
KR101630814B1 (en) SNP molecular markers associated with distinction of grape understock variety and uses thereof
CN112760319B (en) Development and application of aegilops tauschii 2Sl chromosome specific molecular marker
EP3772909A1 (en) Genes associated with resistance to wheat yellow rust
CN111218521A (en) Functional specificity molecular marker of rice blast resistance gene Pib and detection method and application thereof
CN115786564A (en) Rice Pi-ta and Ptr dominant functional molecular marker and application thereof
CN112410463B (en) Molecular marker for resisting bacterial wilt of tomatoes and application of molecular marker
Bolek et al. Molecular breeding of cotton
CN107354151A (en) STR molecular labelings and its application based on the exploitation of sika deer full-length genome
CN108950057B (en) Development and application of Ular pattern wheat powdery mildew resistance gene Pm60 specific molecular marker
CN102094091B (en) Method for separating and detecting spontaneous mutation gene based on agarose gel denaturation and renaturation and biotin affinity adsorption

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
GR01 Patent grant
GR01 Patent grant